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Volume: 15 Issue: 5 October 2017

FULL TEXT

ARTICLE
Immunohistopathologic Characterization of Plasma Cell-Rich Acute Rejection in Living-Related Renal Transplant Recipients

Objectives: Our aim was to analyze the immuno­histopathologic features of plasma cell-rich acute rejection in a living-related renal transplant setting.

Materials and Methods: Renal allograft biopsies of 50 cases of plasma cell-rich acute rejection were reviewed, and the main immunohistopathologic features were analyzed. The biopsies were studied using light microscopy, immunofluorescence, and immunohistochemistry and reported according to Banff classification. Biopsy findings were correlated with graft function and outcome.

Results: From February 2012 to December 2013, 50/1630 (3%) dysfunctional renal allograft biopsies showed plasma cell-rich acute rejection. Among acute changes, interstitial inflammation was of moderate degree in 8 cases (16%) and severe in 42 cases (84%). Mild tubulitis was found in 4 cases (8%), moderate tubulitis in 8 cases (16%), and severe tubulitis in 38 cases (76%). Glomerulitis was found in 2 cases (4%). No presence of arteritis was found. All plasma cell-rich acute rejection cases were of tubulointerstitial type, and most were of type IB. The mean percent of plasma cells on light microscopy in all cases was 28.8 ± 11.7%, and the range was 10% to 60%, with 46 cases (92%) showing plasma cell percent of ≥ 20%. The mean plasma cell percent on immunohistochemistry for CD138 was 29.0 ± 12.4%. Microvascular inflammation was found in 34 cases (68%). C4d testing was done by immuno­fluorescence in 22 cases (44%) and was positive in 8 cases (36%). Interstitial fibrosis/tubular atrophy was mild in 18 (36%), moderate in 28 (56%), and severe in 4 cases (8%). Plasma cell enrichment did not correlate with a variety of clinical and pathologic features (all P > .05).

Conclusions: Plasma cell enrichment is not an inde­pendent prognostic morphologic feature and may represent either T-cell-mediated or antibody-mediated rejection or a mixture of these processes. Further investigations regarding its pathogenesis, accurate categorization, and treatment are needed.


Key words : Allograft biopsy, Graft dysfunction, Kidney transplant

Introduction

Renal transplant is the most effective treatment for patients with end-stage renal disease from a variety of causes.1 Despite improvements in immunosup­pressive drugs and treatment regimens, rejection still remains a formidable challenge, especially for long-term renal allograft survival.2 Rejection processes have traditionally been classified on the basis of rapidity of development of the inflammatory process after transplant. This classification correlated somewhat with the pathogenetic and pathologic features of rejection. However, the latter features were not used for classification purposes until recently.3 Banff classification was developed in an effort to standardize the reporting and classification of rejection on renal allograft biopsies.4-6 Recent updates of Banff classification have tried to move from pathologic to pathogenetic classification of the alloimmune response in kidney transplant.3,7-10 The composition of cellular infiltrates in rejection varies from biopsy to biopsy but is predominantly composed of lymphocytes and macrophages; eosinophils, neutrophils, plasma cells, and mast cells comprise a minor component.3,6 The significance of nonconventional cell infiltrates has been investigated with variable results. Among these, plasma cell enrichment of interstitial cellular infiltrates has received marked attention in recent years.11-18 Many studies have found plasma cell-rich acute rejection (PCAR) to indicate poor prognosis.11-15,19,20 We have previously reported on the treatment and short-term clinical outcomes of a cohort of renal transplant patients with PCAR. With aggressive therapy, short-term results showed a favorable response.21

In this study, we aimed to characterize in detail various histopathologic features of PCAR and to correlate these with other prognostically relevant morphologic, clinical, and outcome variables.

Materials and Methods

We analyzed in detail renal allograft biopsies from 50 patients with PCAR over an 18-month study period (from February 2012 to December 2013). These formed 3% of 1630 renal allograft biopsies performed for unexplained renal allograft dysfunction in 4132 renal transplant recipients on regular follow-up at our center. Graft dysfunction was defined as a rise in serum creatinine level above 20% of baseline value. All biopsies were percutaneous needle core biopsies, and no explanted kidneys were included. All transplants were first transplants, and donors were living related. The research was conducted according to the tenets of the Declaration of Helsinki.

The main demographic, clinical, and immuno­suppressive data of our cohort have been previously published.19 Only selected biopsies were included for this analysis: these included the first biopsy showing plasma cell enrichment (index biopsy) and follow-up biopsies in these cases, if performed. For routine light microscopic studies, all graft biopsies were fixed in 10% neutral buffered formalin and embedded in paraffin. Serial 3- to 4-μm sections were cut and stained with hematoxylin and eosin, periodic acid-Schiff, and silver stains as recommended by Banff classification.

All biopsies were examined initially by light microscopy. Immunohistochemistry was performed for T and B cell markers, CD138, kappa and lambda light chains, and immunoglobulins G, A, and M. In addition, immunofluorescence was performed for C4d in cases with transplant glomerulopathy, and renal panel immunofluorescence was performed in cases with proteinuria > 1 g/24 hours and those with transplant glomerulopathy to confirm or exclude immune complexes.

Statistical analyses
The data were entered into and analyzed by Statistical Package for Social Sciences version 10.0 (SPSS Inc., Chicago, IL, USA). Mean ± standard deviation was used for quantitative variables, whereas qualitative data were given in numbers (percent).

We used chi-square test or Fisher exact test (where applicable) for categorical variables, whereas con­tinuous variables were assessed by independent and paired t test. Pearson or Spearmen correlation analyses were also conducted. P < .05 was considered significant.

Results

The study was conducted from February 2012 to December 2013. During this period, 1630 dysfunc­tional renal allograft biopsies were performed in 4132 patients. Among these biopsies, 50 (3%) showed PCAR, and these constituted the study population for the present report. The demographic, clinical, and laboratory parameters of these 50 patients have already been published.19 The present study was focused on determining the histopathologic characteristics of PCAR and their correlation with graft functional and outcome data.

The index biopsies from 50 patients were analyzed in detail by light microscopy and immuno­histochemistry. All biopsies met the adequacy criteria of Banff classification and included both cortex and medulla in 36 cases (72%) and only cortex in 14 cases (28%). A large percent of biopsies consisted of 2 cores (42: 84%), whereas only 1 core was obtained for 3 cases (6%) and 3 cores were obtained for 5 cases (10%). The mean number of glomeruli per biopsy was 9.2 ± 4.5 (range, 1-16). Global glomerulosclerosis was seen in 14 biopsies (28%) and segmental glomerulosclerosis in 2 biopsies (4%).

Regarding acute changes on graft biopsies, interstitial inflammation was at least of moderate degree in 8 cases (16%) and of severe degree in 42 cases (84%). Regarding the degree of tubulitis, mild tubulitis was found in 4 (8%), moderate tubulitis in 8 (16%), and severe tubulitis in 38 cases (76%). Glomerulitis was found in only 2 cases (4%). No arteritis was found.

Regarding the histologic type of acute rejection, all PCARs were of type I or tubulointerstitial type. No cases of PCAR showed vascular involvement. Among type I, most were type IB (Table 1). The mean percent of plasma cells on light microscopy was 28.8 ± 11.71%. The mean percent of plasma cells on immunohistochemistry for CD138 improved slightly to 29 ± 12.5%.

Morphologic features of microvascular inflam­mation were found in 34 cases (68%). Microvascular inflammation was positive in 24/32 cases (75%) with positive donor-specific antibodies (DSAs) and in 10/18 cases (55.6%) with no DSA. Mean micro­circulation inflammation score was also higher in the DSA-positive group (1.53 ± 1.16) than in the DSA-negative group (0.75 ± 0.86). However, there was no significant difference in the mean microcirculation inflammation values between DSA-positive and DSA-negative groups. The semiquantitative scores of individual lesions of acute rejection are shown in Table 2. C4d was done on fresh frozen tissue by immunofluorescence in 22 cases and was found to be positive in 8 cases (36%).

The mean percent of plasma cells on light microscopy in all cases of PCAR was 28.8 ± 11.7%, and the range was 10% to 60% (Figure 1). Among all cases, 46 (92%) showed plasma cell percent of ≥ 20% of the interstitial infiltrate. The mean plasma cell percent on immunohistochemistry for CD138 was 29.0 ± 12.4%. It is obvious that a good correlation existed between light microscopy and immuno­histochemistry results for quantification of plasma cell percent on the graft biopsies. B cells were also found in variable numbers in 45 cases (90%). The mean percent of B cells was 19.4 ± 12.6%, with range of 5% to 50% (Figure 2A and 2B). All plasma cells were polyclonal, as shown by positivity of these cells for both kappa and lambda light chains (Figure 2C and 2D).

Regarding chronic changes, these were found in nearly all cases. Interstitial fibrosis/tubular atrophy was mild in 18 (36%), moderate in 28 (56%), and severe in 4 cases (8%); scores of individual lesions of chronic changes are shown in Table 2.

No viral inclusions of cytomegalovirus and polyoma virus infection were detected. No immature plasma cells or expansile, destructive nodules of plasma cell infiltrates were noted.

On correlation of plasma cell infiltration and various clinicopathologic characteristics, there was no significant correlation between plasma cell percent and recipient age (P = .24), donor age (P = .26), HLA match (P = .39), interstitial inflammation (P = .30), tubulitis (P = .74), glomerulitis (P = .27), interstitial edema (P = .28), and peritubular capillaritis (P = .10). However, a significant positive correlation was shown with CD138-positive cell counts on immuno­histochemistry (P < .001) and significant negative correlation with CD20-positive cell infiltrates on biopsy (P = .004). There was no correlation with plasma cell infiltration and graft failure rate (P = .30) and graft function at the time of biopsy (P = .34).

Discussion

The focus of renal allograft pathology has typically been on describing the quantity of inflammatory cell infiltrates and their location within the graft. However, the quality of the infiltrate is also equally important. Traditionally, cellular rejection is thought of as “T lymphocyte”-mediated with an admixture of macrophages. The humeral rejection, in contrast, is mediated mostly by neutrophils, although also by lymphocytes and macrophages. Plasma cell infiltration is typically sparse. However, in some settings, plasma cells constitute a significant component of the interstitial infiltrate. Such cases have been labeled as PCAR. The definition of plasma cell enrichment has varied among studies. Most studies have used a 10% cut-off value for labeling a case as PCAR. Plasma cell-rich acute rejection has received marked attention in recent years. There are still few studies on this topic, and these have uniformly reported a poor prognosis.

The observed frequency of PCAR has varied from 2% to 14% in previously published studies.11-15 We found a frequency of 3%, which is comparable to a recent study from India (3.14%).16 The varying frequency of PCAR may be related to varying definitions of plasma cell enrichment used for the diagnosis of PCAR, varying study durations, and the inclusion of explants as well as dysfunctional needle graft biopsies.14 We did not include explants in our study. Differences in time periods of analysis and a change in immunosuppressive drugs may also possibly contribute to this varying frequency.14 We did not systematically analyze the trends in the diagnosis of PCAR in our setting over long periods of time, but we have seen a surge in the number of cases in recent years (data not shown).

The average percent of plasma cells has been variously reported in the previous investigations. Gärtner and associates14 reported 30.38%, which is almost similar to our findings. In a study by Meeham and associates,12 the reported percent was 32%. A lower percent of 26.45% was found in a study from Australia.15 Some investigators11 have presented results as mean number of plasma cells per high-power field (870.1/high-power field). These values were significantly and markedly higher than shown for control cases.

Regarding the distribution of plasma cells, these were found in both diffuse distribution and in clusters, as shown in Figure 1. The clustering of plasma cells was found around glomeruli, blood vessels, and at the corticomedullary junction area. Similar distribution patterns have been previously reported by other investigators.14-18

One interesting finding in our study was that all the rejections were of tubulointerstitial type (type I). No vascular rejection was noted. This phenomenon can develop concurrently with the tubulointerstitial rejection or later on during the course of the transplant. Vascular rejection was observed in 46% of patients in the study by Gärtner and associates14 during the study period. The possible reason for noninclusion of vascular rejection in our series may be the small sample size and the fact that we only included index biopsies in these cases. Plasma cell-rich acute rejection cases may develop vascular rejection in subsequent biopsies.14

Plasma cell enrichment did not correlate with many clinical and pathologic features in this study. The only positive correlation was seen with CD138-positive cell counts on immunohistochemistry, which was only marginally better than light microscopy for the detection and quantification of plasma cells in the interstitial infiltrate. The immunohistochemistry for plasma cells was more helpful in delineating the distribution pattern of these cells, as shown in Figure 1. In addition, plasma cell abundance was negatively correlated with B lymphocyte count. It is under­standable that the plasma cells are the terminally differentiated forms of B-cell precursors, and their numbers are usually inversely related.19,20

There was no correlation of the plasma cell enrichment with graft function or graft outcome. The latter has been uniformly poor in all previously published studies.11-18 However, we have previously published our experience of this cohort of 50 patients who were treated aggressively, and we observed a favorable short-term outcome.21 The follow-up period was brief in that study. It will be important to continue observations on this cohort to see the long-term outcome. Interestingly, all of our cases also showed marked chronic changes in the same biopsies, which showed plasma cell enrichment.

Traditionally, PCAR is considered as a variant of T-cell-mediated rejection.21,22 It has not yet been formally included in the Banff classification.22 Recently, some groups have investigated the humoral component of defection in this entity.18,21 Results from these studies show that the antibody component is a frequent finding in this type of rejection. In the presence of plasma cells in the graft biopsy, it is not surprising that these will be producing antibodies directed against the graft. Thus, PCAR represents a prototype of combined T-cell-mediated rejection and antibody-mediated rejection.

There are certain limitations in the present study. We did not quantify T lymphocytes and its subsets, macrophages, and natural killer cells. We could not perform C4d on all graft biopsies. The origin of the study is a single center, and we did not compare this cohort with nonplasma cell-rich acute rejection.

In conclusion, plasma cell enrichment is not an independent prognostic morphologic feature and may represent either T-cell-mediated or antibody-mediated rejection or a mixture of these processes. Further investigations into its pathogenesis and its accurate categorization and treatment are needed.


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Volume : 15
Issue : 5
Pages : 516 - 520
DOI : 10.6002/ect.2016.0188


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From the Departments of Histopathology, Immunology, Clinical Chemistry, and Nephrology, Sindh Institute of Urology and Transplantation, Karachi, Pakistan
Acknowledgements: The authors declare that they have no sources of funding for this study, and they have no conflicts of interest to declare.
Corresponding author: Muhammed Mubarak, Department of Histopathology, Sindh Institute of Urology and Transplantation, Karachi 74200, Pakistan
Phone: +922 199215752
E-mail: drmubaraksiut@yahoo.com