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ORIGINAL ARTICLE Table of Contents   
Year : 2009  |  Volume : 52  |  Issue : 3  |  Page : 345-348
Diagnostic efficacy of C4d immunostaining in the detection of the humoral component of renal allograft rejection and therapeutic implications

1 Department of Pathology, Pinnamaneni Siddhartha Institute of Medical Sciences, Chinoutapalli, Andhra Pradesh, India
2 Department of Pathology, Sri Ramachandra University, Porur - 600 116, India
3 Department of Medicine, Madras Medical Mission, Chennai, India
4 Director, Chennai Transplant Center, Madras Medical Mission, Chennai, India

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Date of Web Publication12-Aug-2009


Context: In the current scenario of renal transplantation, the role of immunological methods in the detection of C4d has emerged as a useful adjunct in the recognition of acute humoral rejection (AHR). Few reports of this nature are available from the Indian context although there are several from the Western literature. Aims: To study the humoral component of renal allograft rejection in patients presenting clinically with graft dysfunction by histopathological detection of polymorphs in the peritubular capillaries and the expression of C4d using immunological techniques, as well as the response of patients to appropriate antirejection therapy. Settings and Design: This study from a tertiary care center reemphasizes the importance of recognition of AHR as a cause of renal allograft dysfunction. Materials and Methods: Percutaneous renal biopsies were obtained from 40 postrenal transplant patients and evaluated for C4d using immunofluorescence and immunohistochemical methods. Statistical a0 nalysis used: SPSS software. Results: Positive expression of C4d was seen in a total of 19/40 cases (44.4%) indicating immunological evidence of AHR. Diffusely positive cases were treated with IV immunoglobulin therapy, plasmapheresis and Rituximab following which graft function was restored. Patients with minimal to focal positive expression of C4d responded well to pulse steroids and change in immunosuppressive therapy. Conclusions: C4d staining is a useful adjunct to routine histopathological methods in evaluating the humoral component of acute renal allograft dysfunction and helps in planning appropriate antirejection therapy with the goal of achieving long-term graft survival.

Keywords: Acute humoral rejection, antihumoral therapy, C4d

How to cite this article:
Guduru SL, Kuruvilla S, Abraham G, Mathew M, Saravanan S. Diagnostic efficacy of C4d immunostaining in the detection of the humoral component of renal allograft rejection and therapeutic implications. Indian J Pathol Microbiol 2009;52:345-8

How to cite this URL:
Guduru SL, Kuruvilla S, Abraham G, Mathew M, Saravanan S. Diagnostic efficacy of C4d immunostaining in the detection of the humoral component of renal allograft rejection and therapeutic implications. Indian J Pathol Microbiol [serial online] 2009 [cited 2021 May 6];52:345-8. Available from: https://www.ijpmonline.org/text.asp?2009/52/3/345/54990

   Introduction Top

Despite the emergence of molecular techniques, the renal allograft biopsy remains the gold standard for the diagnosis of rejection [1] with a high sensitivity rate [2] and its distinction from other causes of allograft dysfunction.

Acute humoral rejection (AHR) usually occurs a few weeks post transplant and is associated with circulating donor antibodies. [3] AHR may coexist with T-cell-mediated injury (acute cellular rejection -ACR), [4] often contributes to graft injury and is often prognostically worse. Effective therapeutic regimes are available for AHR and if implemented appropriately can result in a favorable long-term graft outcome. With the incorporation of C4d in the recent Banff criteria [5] for AHR, there is a genuine need for documentation of the experience of the diagnosis and therapeutic response of patients from the Indian subcontinent and hence this study was carried out.

   Materials and Methods Top

This study was carried out on percutaneous ultrasound-guided renal biopsies. A total of 40 biopsy samples were included in this study. There were two groups of patients:

Group 1: Sixteen cases had fresh frozen biopsies and these were current cases where the diagnosis was required by the clinician in order to implement appropriate therapy. These biopsies were subjected to immunofluorescence staining using rabbit anti-human C4d polyclonal antibody developed by Regele et al . [6] (mfd by Biomedica, Austria), along with Fitc labeled sheep anti-rabbit antibody (mfd by Serotec U.K) in a three-step indirect immunofluorescence method. Sections were scored for C4d using Olympus BX60 vertical illumination fluorescence microscope.

Group 2: Twenty-four cases were taken from archival material from the Department of Pathology case records (which included referral cases) where retrospective analysis raised a suspicion of AHR. These were formalin-fixed and paraffin-embedded and the sections were subjected to immunohistochemical staining using C4d as the primary antibody using the modified LSAB (labeled streptavidin biotin) technique with 3'-Diaminobenzidine tetrahydrochloride (DAB) as chromogen. The degree of staining was graded as minimal, focal and diffuse, where diffuse had positive staining of >50% of the peritubular capillaries, minimal, when occasional peritubular capillary showed immunoreactivity and focal when <50% of the peritubular capillaries showed positive staining.

The section was deemed unsatisfactory if it contained less than seven glomeruli and no arteries, marginal, if it contained seven glomeruli with one artery and adequate if it had 10 or more glomeruli with at least two arteries.

   Results Top

Renal biopsies were obtained from transplant recipients who were suspected to have acute allograft rejection as the cause of graft dysfunction. The patients in this study ranged in age from 18 to 67 years (mean age-42.5years) and 19 patients were in the 30-49 years age group (mean age-39.5 years). There were 28 males and 12 females. The pre-existing diseases leading to end stage renal disease (ESRD) in this patient group are given in [Table 1].

Histopathologic findings

In patients presenting with graft dysfunction, acute tubular necrosis, polymorphonuclear leucocytes in peritubular capillaries and arterial fibrinoid change [Figure 1] were the parameters evaluated on H and E sections to arrive at the diagnosis/suspicion of antibody-mediated rejection.

The total number of cases studied and the results of immunofluorescence (IF) and immunohistochemical (IHC) staining and the degree of staining are given in [Table 2].

In Group 1, of the 16 cases stained by the immunofluorescence method eight were positive for peritubular capillary C4d staining.

Intense staining for C4d in the form of small round to oval elongated ring-like fluorescence profiles of peritubular capillaries [Figure 2] was readily evident between the larger negative tubular cross-sections in the positive cases. The capillary staining was crisp, linear and continuous, but also had a finely granular pattern at high power, which extended into the lumen from the more linear deposits.

The medullary vessels had less intense C4d deposition compared with the cortex. Arteries with fibrinoid necrosis in a case of hyperacute rejection showed C4d deposition in the tunica media. Bright linear glomerular basement membrane positivity and broad deposits in the glomerular mesangium were seen in positive as well as negative cases. Tubular basement membranes were negative.

The post-transplant cases presenting with graft dysfunction and showing diffuse positivity for peritubular capillary C4d staining underwent plasmapheresis four times and received a course of anti-CD 20 antibody Rituximab 500 mg IV as a single dose and immunoglobulin 1.5 g IV for three days. Following this, standard baseline immunosuppression was continued. Graft function was restored. Cases showing minimal and focal positivity were given regular baseline immununosuppression and was continued with a switchover between Calcineurin inhibitor and MTOR (mammalian target of Rapamycin), inhibitor Rapamycin or Everolimus. These patients responded favourably to this switchover. MTOR inhibitors are also efficacious, particularly in managing antibody-mediated rejection. Twelve patients showed coexistence of cellular rejection. The association of C4d positivity with the morphologic evidence of antibody-mediated rejection was statistically significant ( P 0.001). The sensitivity was 93.47% and specificity 76.47%.

The 24 cases from Group 2 presenting with graft dysfunction and which on review raised a suspicion of AHR were stained by immunohistochemical method. C4d deposits were seen as brown fuzzy rings in the peritubular capillaries [Figure 3]. Thus 11/24 cases were positive for C4d in the peritubular capillaries in this group. This group was studied purely for academic reasons to confirm the clinical suspicion of AHR on retrospective analysis and these patients were not available for treatment.

   Discussion Top

The morphologic confirmation of antibody-mediated rejection in renal biopsies is critical to the diagnosis and management of graft dysfunction in transplanted patients. [7]

Criteria for acute antibody-mediated rejection in renal allografts [8] are:

  1. Morphologic evidence of acute tissue injury, such as acute tubular necrosis, neutrophils in peritubular capillaries or capillary thrombosis, intimal arteritis /fibrinoid necrosis in arteries.
  2. Immunopathologic evidence of antibody action such as C4d in peritubular capillaries. [9]
  3. Serologic evidence of circulating antibodies to donor HLA or other anti-donor endothelial antigens.
Biopsies from patients presenting with graft dysfunction and showing neutrophils in peritubular capillaries and/or tubular necrosis, or arterial fibrinoid necrosis were subjected to C4d staining. Nineteen out of forty biopsies (44.4%) showed bright linear extensive staining for C4d in cortical capillaries and were considered positive for antibody-mediated rejection. The graft function in the Group 1 patients with diffuse positivity, was restored with appropriate anti-humoral therapy. Thus the presence of C4d appears to be a sensitive indicator of anti-donor antibodies. C4d positivity compares favorably with other diagnostic criteria of AHR. [10] Previously, no single pathologic feature had been recognized which consistently distinguished patients with circulating antibodies. The lesions that favored AHR over acute cellular rejection (ACR) were the presence of neutrophils in peritubular capillaries, thrombi and infarction. Of these we found peritubular neutrophils to be the most useful criterion and these were present in all our cases with AHR. A significant number of neutrophils may also occasionally be observed in peritubular capillaries in acute cellular rejection and in Calcineurin inhibitor toxicity 3 loc cit . One patient showed negative staining for C4d despite the presence of neutrophils in peritubular capillaries and on evaluation was found to be on a high dose of cyclosporine. Graft function improved with change of immunosuppression without any anti- humoral therapy. Fibrinoid necrosis of arteries is also commonly regarded as an indicator of humoral rejection and was present in two of our AHR cases.

Cases of graft dysfunction showing mild and moderate C4d positivity of Group 1 were effectively managed with pulse dose of steroids and change in immunosuppression. Specific anti-humoral therapy in the form of plasmapheresis and anti CD-20 antibody was required only in the diffusely positive cases. This suggests that C4d staining not only indicates humoral rejection responses but also aids in planning appropriate anti-rejection management [11] which is very essential in achieving long-term graft survival. One case of chronic allograft injury with diffuse positivity for C4d showed restoration of graft function with plasmapheresis and anti-CD 20 antibody. This is consistent with the previous studies, [12],[13],[14] emphasizing the role of humoral responses in the pathogenesis of chronic allograft injury. [15] Thirteen out of the nineteen cases showed coexisting cellular rejection.

We conclude from the data presented here that in severe graft dysfunction humoral immune mechanisms should be considered, even when histopathologic features of humoral rejection are equivocal. C4d deposition in peritubular capillaries seems to be a reliable marker of humoral alloreactivity. Its detection might therefore be simple and inexpensive and can help identify patients who are likely to benefit from anti-humoral therapy. [16]

To summarize, cases of graft dysfunction with diffuse positivity for C4d staining require specific anti-humoral therapy. Cases presenting with minimal and focal positivity for C4d showed improved graft function with pulse dose steroids and change in immunosuppression. C4d detection is therefore a useful adjunct in the diagnosis of humoral responses in renal transplant patients presenting with graft dysfunction and helps in guiding clinical decisions for achieving long-term graft survival.

   Acknowledgements Top

The authors would like to acknowledge the partial financial aid given by the Indian Council of Medical Research. (ICMR). The partial financial support and encouragement given by the Tamil Nadu Kidney Education and Research (TANKER) foundation is gratefully acknowledged by the authors.

   References Top

1.Racusen LC, Solez K, Colvin RB, Bonsib SM, Castro MC, Cavallo T, et al . The Banff '97 working classification of renal allograft pathology. Kidney Int 1999;55:713-23.  Back to cited text no. 1  [PUBMED]  [FULLTEXT]
2.Colvin RB, Cohen AH, Saiontz C, Bonsib S, Buick M, Burke B, et al . Evaluation of Pathological criteria for acute renal allograft rejection: Reproducibility, Sensitivity and clinical correlation. J Am Soc Nephrol 1997;8:1930-41.  Back to cited text no. 2  [PUBMED]  [FULLTEXT]
3.Collins AB, Schneeberger EE, Pascual MA, Saidman SL, Williams WW, Tolkoff-Rubin N, et al . Complement activation in acute humoral renal allograft rejection. J Am Soc Nephrol 1999;10:2208-14.  Back to cited text no. 3  [PUBMED]  [FULLTEXT]
4.Crespo M, Pascual M, Tolkoff-Rubin N, Mauiyyedi S, Collins AB, Fitzpatrick D, et al . Acute humoral rejection in renal allograft recipients: Incidence, serology and clinical characteristics. Transplantation 2001;71:652-8.  Back to cited text no. 4  [PUBMED]  [FULLTEXT]
5.Trpkov K, Campbell P. Pazderka F, Cockfield S, Solez K, Halloran PF. Pathologic features of acute renal allograft rejection associated with donor-specific antibody analysis using the Banff grading schema. Transplantation 1996;61:1586-92.  Back to cited text no. 5    
6.Regele H, Exner M, Watschinger B, Wenter C, Wahrmann M, Osterreicher C, et al . Endothelial C4d deposition is associated with inferior kidney allograft outcome independently of cellular rejection. Nephrol Dial Transplant 2001;16:2058-66.  Back to cited text no. 6  [PUBMED]  [FULLTEXT]
7.Truong LD, Barrios R, Adrogue HE, Gaber LW. Acute antibody-mediated rejection of renal transplant: Pathogenetic and diagnostic considerations. Arch Path Lab Med 2007;131:1200-8.  Back to cited text no. 7  [PUBMED]  [FULLTEXT]
8.Tanaka T, Kyo M, Kokado Y, Takahara S, Hatori M, Suzuki K, et al . Correlation between the Banff 97 classification of renal allograft biopsies and clinical outcome. Transpl Int 2004;17:59-64.  Back to cited text no. 8  [PUBMED]  [FULLTEXT]
9.Racusen LC, Colvin RB, Solez K, Mihatsch MJ, Halloran PF, Campbell PM, et al . Antibody mediated rejection criteria -an addition to the Banff's '97 classification of Renal allograft rejection. Amer J Transplant 2003;3:708-14.  Back to cited text no. 9    
10.Bates WD, Davies DR, Welsh K, Gray DW, Fuggle SV, Morris PJ. An evaluation of the Banff classification of early renal allograft biopsies and correlation with outcome. Nephrol Dial Transplant 1999;14:2364-9.  Back to cited text no. 10  [PUBMED]  [FULLTEXT]
11.Becker YT, Becker BN, Pirsch JD, Sollinger HW. Rituximab as treatment for refractory kidney transplant rejection. Am J Transplant 2004;4:996-1001.  Back to cited text no. 11  [PUBMED]  [FULLTEXT]
12.Montgomery RA, Zachary AA, Racusen LC, Leffell MS, King KE, Burdick J, et al . Plasmapheresis and intravenous immune globulin provides effective rescue therapy for refractory humoral rejection and allows kidney to be successfully transplanted into cross-match positive recipients. Transplantation 2000;70:887-95.  Back to cited text no. 12  [PUBMED]  [FULLTEXT]
13.Hara S, Matsushita H, Yamaguchi Y, Kawaminami K, Horita S, Furusawa M. Allograft glomerulitis: Histologic characteristics to detect chronic humoral rejection. Transplant Proc 2005;37:714-6.  Back to cited text no. 13  [PUBMED]  [FULLTEXT]
14.Joosten SA, Sijpkens YW, van Kooten C, Paul LC. Chronic renal allograft rejection: Pathophysiologic considerations Kidney Int 2005:68:1-13.  Back to cited text no. 14    
15.Vadivel N, Tullius SG, Chandraker A. Chronic renal allograft nephropathy. Semin Nephrol 2007;27:414-29.  Back to cited text no. 15  [PUBMED]  [FULLTEXT]
16.Faguer S, Kamar N, Guilbeaud-Frugier C, Fort M, Modesto A, Mari A, et al . Rituximab therapy for acute humoral rejection after kidney transplantation. Transplantation 2007;83:1277-80.  Back to cited text no. 16  [PUBMED]  [FULLTEXT]

Correspondence Address:
Sarah Kuruvilla
Department of Pathology, Sri Ramachandra University, Porur, Chennai - 600 116
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.54990

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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2]

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