Background: Delayed graft function (DGF) immediately after kidney transplantation is considered a risk factor for acute rejection. According to clinical guidelines, a weekly allograft biopsy should be performed until DGF resolves. Based on clinical evidence, the first biopsy is considered appropriate. However, the recommendation for further biopsies is based on sparse evidence from era of earlier immunosuppression protocols, and the benefit of the second and further biopsies remains uncertain. The aim of this study was to reevaluate this policy. Methods: The database of a transplant medical center was retrospectively reviewed for all patients who underwent kidney transplantation in 2011–2020. Those with DGF who performed two or more graft biopsies within the first 60 days after transplantation were identified. Clinical data were collected from the medical files. The rates of diagnosis of acute rejection at the second and subsequent biopsies were analyzed relative to the previous ones. Results: Kidney transplantation was performed in 1,722 patients during the study period, of whom 225 (13.07%) underwent a total of 351 graft biopsies within 60 days after transplantation, mostly due to DGF. A second biopsy was performed in 32 patients (14.2%), and a third biopsy in 8, at weekly intervals. In 2 patients (6.25%), the diagnosis changed from the first biopsy (acute tubular necrosis or toxic damage) to acute rejection in the second biopsy. In both, the rejection was borderline. Third and fourth biopsies did not add information to the previous diagnosis. Conclusions: The common practice of performing sequential biopsies during a postoperative course of DGF seems to be of low benefit and should be considered on a case-by-case basis.

© 2022 The Author(s). Published by S. Karger AG, Basel

Introduction

The early postoperative course after kidney transplantation is often complicated by a period of delayed graft function (DGF). The causes of DGF are acute tubular necrosis (ATN), acute rejection, microbial and viral infection, thrombotic microangiopathy, recurrent primary renal disease, and drug intoxication. Most of these conditions are treatable and reversible. However, DGF poses a risk of acute rejection [1-3], and DGF and acute rejection together are associated with worsened long-term outcomes including irreversible kidney damage and even graft loss. Thus, early detection of acute rejection during the DGF period is of prime importance. Biopsy of the transplanted kidney is the gold standard for diagnosis of acute rejection; there are no noninvasive tests (urine, blood, or imaging) that are sufficiently reliable. According to the guidelines of the Kidney Disease Improving Global Outcomes (KDIGO) Transplant Work Group [4], published in 2009, “it is… prudent to obtain periodic biopsies of the kidney during DGF to diagnose acute rejection” [p. S31]. Our center follows this recommendation and performs protocol biopsies every 7–10 days until DGF resolves or primary graft nonfunction is diagnosed [5].

The role of the first biopsy in DGF is clear as the chances of diagnosing acute rejection at the first biopsy during DGF are relatively high [5]. The need for a second biopsy to detect acute rejection was first suggested in 2002 in a study by Qureshi et al. [6] upon 410 transplant recipients. Of the 134 patients with DGF, 65 (62.5%) underwent a protocol biopsy every 7–10 days. Acute rejection was detected in 33 (50.8%): 24 in the first biopsy, 8 in the second, and 1 in the third. On Kaplan-Meier analysis, the cumulative incidence of acute rejection in the first 40 days after transplantation was 57.2%. This study was the basis for the KDIGO recommendation mentioned above [4].

However, in the recent years, early (within 6 months of transplantation) acute rejection rates have dropped significantly thanks to advances in the understanding of the histopathology of transplantation, improvements in the grading system of kidney transplant biopsies (Banff Classification), and the introduction of new immunosuppressive regimens [7, 8]. This is especially true for nonsensitized patients receiving a standard-criteria donation after brain death [9] or living donation. The present study was prompted by these findings, together with the sparsity of relevant publications in the literature for almost two decades. We assumed that during DGF the first biopsy is critical and in this study we focused on examining the role of the second and further biopsies.

Patients and MethodsStudy Design

The study was approved by the Institutional Review Board of Rabin Medical Center. The healthcare database of Rabin Medical Center was retrospectively reviewed for all patients who underwent kidney transplantation (from either living or deceased donors) between 2011 and 2020 and were subsequently diagnosed with DGF. The study cohort consisted of patients among this group who underwent two or more biopsies during the DGF period, within 60 days after transplantation.

DGF was defined as the need for dialysis at least once after transplantation (not including the first 24 h). If DGF persisted more than 7–10 days, a weekly allograft biopsy was performed until dialysis was discontinued.

Biopsies were performed by a radiologist using needle aspiration under sonographic guidance. Three to four rolls of tissue were taken in each case. The fresh tissue was immediately transferred to the hospital’s pathology laboratory and processed on the same or the next day. Direct immunofluorescence was performed routinely, including C4d staining.

All biopsy samples were examined by an expert in transplant pathology. For purposes of this study, the biopsy reports were revised and classified according to the 2018 update of the Banff Classification criteria. For the sake of simplicity, when another diagnosis is made on top of rejection, we report here rejection only. The donor-specific antibody test was performed in most patients concurrent with the biopsy study, and the results served to support or refute the diagnosis of active antibody-mediated rejection (AMR) (Banff Classification, category 2). The following clinical variables were collected from the medical files: recipient and donor demographic data (sex, age), etiology of the native renal disease, type and duration of pre-transplant dialysis, primary versus repeated transplantation, number of HLA-A, B, and DR mismatches between donor and recipient, immediate pre-transplant panel-reactive anti-HLA antibody level, type of donor (living or deceased), cold ischemia time, and type of induction therapy (basiliximab or rabbit anti-thymocyte globlulin).

Immunosuppression

Induction therapy consisted of either basiliximab (Simulect®; Novartis, Basel, Switzerland) 20 mg on days 0 and 3 or rabbit anti-thymocyte globlulin(Thymoglobulin®; Genzyme, Cambridge, MA, USA) 3 mg/kg total dose on 3 separated days (dose given when lymphocyte count was higher than 100 cells/mL). Patients were also given intravenous methylprednisolone, 500 mg on day 0, 250 mg on day 1, and 125 mg on day 2. As maintenance, a triple-agent calcineurin-inhibitor-based regimen was administered. From day 0, patients received oral tacrolimus (Adoport®; Novartis) 0.15 mg/kg/day or cyclosporine (Neoral®; Novartis) 7 mg/kg/day, and mycophenolic acid (Myfortic; Novartis®) 1,440 mg/day. The tacrolimus dose was adjusted to achieve a trough level of 8–15 ng/mL during the first 3 months and 6–8 ng/mL thereafter; the cyclosporine dose was adjusted to achieve a trough level of 200 ng/mL during the first month and 100–150 ng/mL thereafter. From day 3, patients received oral prednisone 20 mg/day, gradually tapered down to 5 mg/day after 3 months.

Concomitant Medications

Patients were given prophylactic treatment for Pneumocystis jirovecii infection (oral trimethoprim/sulfamethoxazole 80 + 400 mg daily for 3 months) and cytomegalovirus (oral valganciclovir 450 mg daily for 3 months, regardless of the risk). A proton pump inhibitor and aspirin were also administered for 3 for months after transplantation.

Statistical Analysis

Data are presented as mean and standard deviation, count, and/or percentage. For comparison between groups, the independent two-sided t test was used for numerical variables and χ2 for dichotomous variables. Statistical significance was set at p < 0.05.

Results

During the study period, 1,722 patients underwent kidney transplantation at our tertiary hospital. DGF was diagnosed in 515 (29.9%), of whom 225 underwent a total of 351 graft biopsies within 60 days after transplantation, most due to DGF and the rest due to early deterioration of a functioning graft. The type of donor in this group of patients with DGF was donation after brain death in 90.1% of cases, and the rest are donations from a living donor. In Israel, due to ethical issues, just in the last 3 years there are donations after uncontrolled cardiocirculatory death (DCD), while controlled DCD are not permitted. In addition, the rate of kidney transplantation from DCD in our center is negligible with less than 1% in the last years. Among 515 patients with DGF, 186 underwent only one biopsy with the following results: ATN (Banff Classification, category 6) in 102 cases (45.3%), T-cell-mediated rejection (TCMR; Banff Classification, category 4) in 58 cases (25.7%), AMR (Banff Classification, category 2) in 18 cases (8.0%), and other diagnoses in 47 cases. On average, the first biopsy was performed on postoperative day 11. Among 76 cases of acute rejection, panel-reactive anti-HLA antibody (either I or II) was higher than 3% in 15 cases.

Thirty-nine patients had two or more biopsies, for a total of 87 biopsies. Seven out of 39 patients were excluded from the present analysis for the following reasons. In 2 patients, the first biopsy was done during the transplant surgery itself because of an unfavorable appearance of the kidney, although blood supply and drainage were adequate. In 2 patients, after the first biopsy and adequate treatment, kidney function initially improved but then deteriorated, which prompted a second biopsy. It was decided not to consider this as prolonged DGF. In 1 patient, the material of the first biopsy was insufficient to make a diagnosis, so a second was performed. In 2 patients, a second biopsy was done despite an improvement in renal function.

The clinical characteristics of the remaining 32 patients are shown in Table 1. ATN was diagnosed in the first biopsy in 19 patients (59%), TCMR in 4 patients (13%), AMR in 6 patients (19%), and “other” in 3 patients (9%, 2 patients with wide necrosis and 1 patient with toxic damage). In 27 patients (84%), the diagnosis in the second biopsy was the same as in the first. In 2 patients, the diagnosis changed from ATN or toxic damage to borderline TCMR (Banff Classification, category 3). In the other 3, the diagnosis changed from AMR to ATN, TCMR to ATN, and ATN to “other.” Among the 4 patients with TCMR on the first biopsy, 1 had grade IB, 1 had grade IIA, and 2 patients had grade IIB TCMR. A resolution was noted on subsequent biopsies in all four of them. Figure 1 elaborates the change in diagnosis from the first to the second biopsy and from the second biopsy to the final outcome.

Table 1.

Clinical characteristics of 32 patients with DGF who underwent 2 or more biopsies within the first 60 days after kidney transplantation

Fig. 1.

Changes in diagnosis from the first to the second biopsy in 32 patients with DGF after kidney transplantation. AMR, antibody-mediated rejection; ATN, acute tubular necrosis; TCMR, T-cell-mediated rejection.

In 8 patients, a third biopsy was performed. The diagnosis was changed in 2 patients, from “other” to ATN and from borderline TCMR to ATN. In 1 patient, 4 serial biopsies were performed, all showing AMR. Overall, the second biopsy commenced further treatment in some way in 17 out of 32 patients (53.1%).

Among the 32 patients with a second biopsy, 15 were found to have primary nonfunction and 13 had DGF for an average duration of 28 days. In the remaining 4 patients, the graft partially functioned immediately but with some compromise that warranted biopsy study. For simplicity reasons of the statistical analysis, these 4 patients were included in the DGF group. The results are shown in Table 2. There were no significant between-group differences. No deaths occurred during a follow-up time of 3 months in the group of 32 patients with two or more biopsies after kidney transplantation.

Table 2.

Comparison of clinical characteristics of patients with a final diagnosis of PNF or DGF

Discussion

We sought to investigate the yield of sequential biopsies in kidney transplant recipients with DGF in the early post-transplant period. The study was conducted in 39 patients treated with contemporary immunosuppressive protocols. The results suggest that the KDIGO recommendation of 2009 to perform weekly biopsies until resolution of DGF or a diagnosis of PFN may be outdated and warrants further evaluation.

Role of the Early First Biopsy during DGF after Kidney Transplantation

Previous studies prove that the importance of the first biopsy during DGF is unequivocal, leading to detection rate of acute rejection that ranges from 5% to 25%. In the first study that highlights the importance of early biopsy during DGF, performed in 2002, Qureshi et al. [6] reported that 32.7% of 410 kidney transplant recipients had DGF, of whom 24.6% were found to have biopsy-proven acute rejection. Two-thirds of patients were diagnosed at the first biopsy and the rest at subsequent biopsies. Accordingly, Puliatti et al. [10] retrospectively evaluated 358 kidney transplant recipients from 2002 to 2006, of whom 101 (28.2%) had DGF and underwent graft biopsy on postoperative day 7. Acute rejection was diagnosed in 19 (18.8%).

Hatoum et al. [11], in a study of 420 kidney transplant recipients from 2008 to 2011, identified 83 (19.8%) who had DGF of 1 week’s duration or more and underwent weekly biopsies until its resolution. Demographically, these patients resembled ours except for a higher rate of deceased donor grafts. Forty-seven patients underwent one graft biopsy, 30 patients – 2, 5 patients – 3, and 1 patient – 4. However, the rate of biopsy-proven clinically significant rejection was only 4.8%, much lower than in our cohort. In 2 of the 4 cases of acute rejection, the diagnosis was made at the first biopsy and in 2, at the second biopsy. The authors concluded that given the low acute rejection rate, it may be reasonable to reduce the frequency of biopsies for DGF and to limit them to a select group of patients at high risk of rejection.

More recently, Favi et al. [7] studied the results of early biopsies performed after kidney transplantation in 270 patients operated from 2010 to 2013. One-third of donations were made after circulatory death and two-thirds after brain death. DGF was recorded in 116 patients (43%). During the first 28 days after transplantation, 98 biopsies were performed in 88 patients. TCMR was diagnosed in 5.1% of biopsied grafts and borderline rejection in 3.1%. No cases of AMR were reported. Rejection was more often associated with worsening graft function or lack of improvement in graft function than with DGF (66.7% vs. 3.5%; p = 0.007 and 33.3% vs. 3.5%; p = 0.014, respectively) and with biopsies performed between day 15 and 28 than between day 0 and 14 (31.2% vs. 3.7%; p < 0.001). The study did not elaborate the results of second and further biopsies compared with the first biopsy.

In our study, the DGF rate was 29.9% which is similar to the results in the mentioned studies above. The diagnosis of acute rejection (TCMR and AMR together) in the first biopsy during DGF in our cohort is 33.7%. These results are relatively high when compared to previous studies [6, 7, 10, 11]. This difference may be related to variation in center practices, patient population, and other factors such as initial dose of T-cell induction therapies. It stands in contrary to our knowledge about the progress in induction protocols. Two decades ago, induction protocols favored high-dose steroids or anti-IL-2 receptor antagonists and tacrolimus and mycophenolic acid were not standard maintenance immunosuppressant [12-15]. Since then, the widespread use of lymphocyte-depleting agents and tacrolimus-mycophenolic acid immunosuppressive regimens has led to a significant reduction in early acute rejection rates [11, 12, 14, 16-18].

Our results add to previous evidence and consolidate the impact of the first graft biopsy. Hence, the first biopsy is suggested to be performed during the first 7–10 postoperative days.

Role of the Second Biopsy during DGF after Kidney Transplantation

Few studies have been conducted upon the yield and necessity of a second biopsy in the setting of DGF following kidney transplantation. In the 2002 study of 410 transplants by Qureshi et al. [6], the DGF rate was 32.7% and acute rejection rate of 50.8%. In 27% of cases, acute rejection was first detected only at the second biopsy. The relatively high rates of both DGF and acute rejection may have justified repeated weekly sampling. By contrast, almost 15 years later, Hatoum et al. [11] found that of 88 patients with DGF after kidney transplantation, only 4 (4.8%) had acute rejection and in only 1 of these patients was the diagnosis made at the second biopsy. The authors concluded that under current immunosuppression protocols, rejection during DGF is uncommon, and the utility of serial biopsies during DGF is limited.

In the present study, the second biopsy led to a change in the primary diagnosis in 5 of 32 cases (15.6%) (Fig. 1). Since undetectable rejection is the main reason for sampling kidney grafts, the yield of the second biopsy in terms of a diagnosis of acute rejection is an important consideration. Only in 2 cases, the diagnosis at the first biopsy (ATN and toxic damage, 1 case each) was changed to borderline TCMR at the second biopsy. None of the third and fourth biopsies revealed acute rejection that had not already been diagnosed at the previous biopsies. Since 17 out of 19 cases of ATN were consistently diagnosed with ATN, it would be safe to say that this diagnosis makes further biopsy redundant. In addition, since 8 out of 10 cases of acute rejection were consistent, some may argue that there is clinical importance to continue to diagnose and give further treatment for persistent rejection.

In conclusion, we examined the benefit of performing two or more consecutive biopsies during DGF after kidney transplantation to exclude rejection. Although the issue has hardly been investigated, a significant clinical guideline was based on this weak evidence. Early biopsy, on postoperative day 7, is necessary to recognize acute rejection, but we believe that the lower rates of rejection with modern protocols of immunosuppression reduce the benefit of weekly biopsies. In centers with high rejection rates that use donors meeting expanded criteria, in patients with high risk of rejection and on a case-by-case basis, a second biopsy should be considered.

Statement of Ethics

This study protocol was reviewed and approved by the Institutional Review Board of Rabin Medical Center, approval number 0109-20-RMC. Informed consent was not necessary due to the retrospective nature of the study. Informed consent was not required to this study (the Institutional Review Board of Rabin Medical Center).

Conflict of Interest Statement

The authors declare no conflict of interest.

Funding Sources

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author Contributions

Ohad Guetta: design, analysis, and interpretation of the data and drafting the article. Anton Osyntsov: study design and revision of the article. Ruth Rahamimov: conception of the study. Ana Tobar, Moshe Israeli, and Yasmin Masarwa: analysis and interpretation of the data. Michael Gurevich: providing intellectual content of critical importance. Vladimir Tennak: study design. Vadym Mezhybovsky and Aviad Gravetz: revision of the article. Sigal Eisner: design and interpretation of the data. Eviatar Nesher: conception and design, analysis, and interpretation of the data and revision of the article.

Data Availability Statement

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding authors.

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