For patients with end-stage renal failure, transplantation remains the treatment of choice. However, despite the ever-increasing success of renal transplantation, graft failure remains a major problem after transplantation (Dallman, 2003). Renal allograft failure can be caused by a large variety of diseases; among them, acute rejection is both frequent and clinically significant (Mauiyyedi and Colvin, 2002). Acute rejection can be mediated by allo-reactive inflammatory cells [acute cellular rejection (ACR)] or specific antibodies [antibody-mediated rejection (AMR)] (Nickeleit and Andreoni, 2007), and the distinction between them is therapeutically important but pathologically difficult (Collins et al., 1999). Acute humoral rejection (AHR) occurs in 20–30% of acute rejection cases with a poorer prognosis than ACR and is refractory to conventional immunosuppressive therapy (Mauiyyedi et al., 2002). Identification of AHR in biopsy specimens has been problematic, because no morphologic features described to are either pathognomonic or universal. Several features are helpful in this regard, including neutrophils in peritubular capillaries (PTCs), fibrinoid necrosis, and glomerular thrombi. Unfortunately, immunofluorescence for IgG, IgM, C3, or fibrin is not as helpful as one might expect, showing no statistically significant difference between AHR and ACR. This traditional view is changed in the work by many authors from many years ago because C4d has led to major changes in our understanding of kidney transplant pathology (Trpkov et al., 1996; Pascual et al., 1998). Complement fixation is strongly associated with the ability of antibody to mediate AMR in humans. Recipients with antidonor HLA class I antibodies able to fix complement showed more severe rejection, as measured by graft loss. Those with noncomplement-fixing antibodies had a prognosis similar to those without antibodies. Complement-fixing HLA class II antibodies did not affect graft survival, even though they were associated with C4d deposition (Wahrmann et al., 2006). C4d is the degradation product of the activated complement factor C4, a component of the classical complement cascade that is typically initiated by binding of antibodies to specific target molecules. Following activation and degradation of the C4 molecule, thioester groups are exposed that allow transient, covalent binding of the degradation product C4d to endothelial cell surfaces and extracellular matrix components of vascular basement membranes near the sites of C4 activation (Bohmig et al., 2002). Fibrin is the end product of the coagulation cascade. Fibrin clots compromise glomerular capillary flow (leading to focal ischemia and necrosis) and attract both macrophages and leukocytes (Hertig and Rondeau, 2004). Fibrin can be found in arteries with fibrinoid necrosis/arteritis and is specific for AMR (Hertig and Rondeau, 2004).
This study aimed to interpret both C4d and fibrin as diagnostic tools for early assessment of AHR.
Patients and methods
This work represents a retrospective selected study on 53 renal allograft biopsies. Needle biopsies had been taken from transplanted patients in the period between January 2005 and May 2008 at the Urology and Nephrology center – Mansoura Faculty of Medicine. The selection of patients depended on the increase in serum creatinine from the basal level maintained during the first 3 months after the onset of transplantation. Clinical data were collected from the database system of the transplant sheet. There were 40 men (75%) and 13 women (25%), ranging in age between 11 and 50 years, with an average age of 28 years (±3.7SD). As for consanguinity, 34 recipient patients out of 53 (64.1%) had related donors, whereas 19 (35.9) had unrelated donors. The original kidney diseases among the recipient patients were as follows: 40 patients (75.5%) had end-stage chronic pyelonephritis, four patients (7.5%) had nephrosclerosis, and nine patients (17%) had membranoproliferative glomerulonephritis. All patients had received their graft for the first time and were maintained on primary triple immunosuppressive therapy in the form of steroids, Cyclosporine, and Azathioprie. Formalin-fixed, paraffin-embedded blocks were cut into 2–3μm thickness and mounted on glass slides and positively charged slides. Alternate slides were stained with hematoxylin and eosin stains, periodic acid- Schiff stain, and Masson trichrome stain as a routine workup following standard procedures for staining (Racusen et al., 1999). Cases of AHR were previously stained for C4d using the immunohistochemical (IHC) technique. The slides were reviewed by pathologists for histological re-examination and re-evaluation of acute rejection according to the revised Banff schema (2007) (Solez et al., 2008).
Sections that were carried on positively charged slides were placed on water. Antigen retrieval was carried out in a preheated oven with a staining dish containing Sodium Citrate, Buffer PH 6, until the temperature reached 95–100°C. The lid was loosely placed on the staining dish containing the slides and incubated for 20–40 min. The oven was turned off, the staining dish was held at room temperature, and the slides were allowed to cool for 20 min. Sections were rinsed in phosphate-buffered saline twice for 2 min.
The pretreated sections were then stained by the monoclonal C4d antibody using the indirect immunofluorescent (IF) method (Quidel Corporations, Santa Clara, California, USA. Cat.A213) at a dilution of 1/100.
Fibrinogen was purchased from Dako Laboratories, Catalogue number F011102, at a dilution of 1/60. Fibrinogen was stored in phosphate buffer containing 15 mmol/l NaN 3, pH 7.2. The sections were placed in water, kept flat in a humidity chamber, and then covered with a thin film of buffer. Drops of protease solution were added to each slide and then kept in a humidity chamber, which was placed in an incubator at 37°C for 10 min. Fibrinogen was incubated for 60 min. A cover slip with Aqua-Mount (Dako laboratories) was used.
Evaluation of slides with immunofluorescent staining
This was done using an Olympus BX60 (Olympus Corporation, Tokyo, Japan) epiillumination fluorescence microscope at X40 for C4d interpretation and at different magnification powers for fibrinogen interpretation.
The percentage of stained tissue on immunofluorescence that had a linear, circumferential staining pattern in PTCs was recorded. The minimal sample for evaluation is 5 high power field of the cortex and/or the medulla without scarring or infarction. Staining should be more than 1+ in intensity. The actual percentage of tissue involved was recorded (diffuse if >50% of the biopsy area affected and focal if >10% and <50% affected).
Fibrin can be seen in small arteries and arterioles with arteritis and fibrinoid necrosis. It can also be detected in glomeruli as fibrin thrombi and also in PTs as thrombi in severe AMR.
All data were analyzed using SPSS for windows software (version 3, IBM, Chicago, IL, USA). C4d detection in the biopsies was correlated to different criteria using univariate analysis. Numerical variables were compared using the Mann–Whitney test, a nonparametric test equivalent to the t-test. Categorical data were compared using the χ2-test or Fisher's exact tests for a small sample size. P values less than or equal to 0.05 were considered significant.
The age distribution and consanguinity of the recipient patients are shown in Tables 1–5 and Graph 1.
In the AHR categories, the fibrin was positive in 64% of the cases. In ACR, fibrin was positive in only 16.7% of the cases; this difference was statistically significant (P<0.05), Tables 6 and 7, Figs 1–5, and Graph 2.
AMR arising in the first few weeks after transplantation is increasingly becoming a challenge in renal allotransplantation. AMR is usually associated with profound allograft dysfunction and inferior allograft survival, and the majority of it occurs due to de-novo production of donor-specific antibodies (Dragun and Rudolph, 2007).
Regarding consanguinity among recipients, (63.6%) out of 22 recipients (77.8%) were negative for C4d. This correlation was not significant (P value=0.3), and this was not consistent with the study carried out. This may be due to the small number of cases studied and also the HLA mismatch between the cases. In terms of the original kidney disease that led to end-stage kidney disease, it was chronic pyelonephritis in 75.5%, nephrosclerosis in 7.5%, and membranoproliferative glomerulonephritis in 17% of the patients. This was statistically nonsignificant (P value=0.3) and consistent with the study carried out by (Poduval et al., 2005). This may be due to the fact that the pathogenesis of AHR is dependent mainly on immunological mechanisms due to auto antibodies to the donor kidney rather than the nonimmunological causes of the listed original kidney diseases that lead to end-stage kidney disease. Regarding the interpretation of C4d staining, this study revealed that (41.5%) of the biopsies were positive for C4d staining by the immunofluorescence technique and these were classified according to the Banff grading system as AHR cases. Thirty-four percent of the cases were classified according to the Banff grading system as ACR and 24.5% cases were classified as others not related to rejection (acute tubular necrosis and acute drug toxicity). The Banff schema defines positive C4d by immunofluorescence as ‘widespread, strong linear and circumferential peritubular capillary staining in cortex or medulla, excluding scar or necrotic areas and involves more than 50% of biopsy area’ (Colvin and Nickeleit, 2006). This pattern has been associated with circulating allo-antibodies. This is why it was detected in capillary endothelial cells, not the arterial ones, as the capillary endothelium expresses both HLA-class I and class II antigens (Troxell et al., 2006). Regarding the correlation of the histological criteria and the Banff grading system to positive C4d IF detection, the results of this study showed that light microscopic changes suggestive of acute tubular cell injury (thinning of the cytoplasm, tubular dilatation, and loss of brush border, cell membrane disruption, apoptosis, and increased proliferation) are frequent. In the present study, 64% of the biopsies showed acute tubular necrosis and 36% of the biopsies showed tubulitis. These (64%) biopsies with AHR showed a histological criteria of acute tubular necrosis (P value=0.011) and these confirmed the results obtained by (Mauiyyedi et al., 2002). The peritubular capillaries (PTCs) are dilated and contain neutrophils in up to 73% of biopsies, but this usually involves a few (one–four) neutrophils in a small number of capillaries (PTC-one), with few showing marked (more than 10) neutrophil predominance (PTC-three). In this study, 73% of 22 biopsies of AHR showed inflamed, congested peritubular capillaries versus 28% of 18 cases diagnosed with ACR. This was statistically significant (P value=0.005) as in the study carried out by (Mauiyyedi et al., 2002). Fibrin can be observed in severe AMR in PTCs as detected by IF staining (nephrectomy specimens) (Cornell et al., 2008). This is mainly due to the activation of complement mechanisms and the resultant neutrophil and macrophage chemotaxis and activation. In this study, this histological picture was detected in 23% of the cases of AMR, which was in accordance with the study carried out by (Nickeleit and Andreoni, 2007). This is due to the fact that complement split products bind to the antigen–antibody complexes and trigger vascular injury with subsequent fluid and cellular extravasations into the interstitial space. Five biopsies also displayed significant interstitial T-cell infiltration ‘with its characteristic deeply basophilic nucleus, scant cytoplasm, and surrounding halo’ and tubulitis, which was in consistent with the study carried out by (Cornell et al., 2008), indicating a coexisting component of cell-mediated rejection as the increased expression or even de-novo expression of some of the HLA class I and class II antigens increases in response to environmental factors, including cytokines, accounting for clinical conditions that promote AMR, as well as the frequent association of AMR and cell-mediated rejection (Nickeleit and Andreoni, 2007; Truong et al., 2007). The small arteries and arterioles showed intimal arteritis, thrombosis, necrosis, or combinations of these changes. Transmural arteritis is seen in rare instances. These changes strongly indicate AMR, but it is statistically insignificant (P value=0.071). The results are in contrast to the study carried out by (Truong et al., 2007), as they are described in both C4d+ve and C4d−ve cases, but with a higher frequency in the former (Ranjan et al., 2008). This is due to the role of CD4 T-helper cells in T-cell-mediated vascular injury in addition to B-cell activation, immunoglobulin production, and subsequent antibody-mediated vascular injury. Fibrin is detected by immunofluorescence in arteries with fibrinoid necrosis/arteritis, and is specific for AMR. Fibrin is rarely seen in peritubular capillaries in grafts with a negative cross match, and it is usually associated with severe AMR (Colvin and Nickeleit, 2006). Concerning the detection of fibrin deposits in arterial fibrinoid necrosis, in this study, high sensitivity (65%) and specificity (78%) were found for diagnosing AHR as in the study carried out by Mauiyyedi et al. (2002). This is because complement activation results in intravascular thrombosis by triggering endothelial synthesis of procoagulants, including tissue factor and subsequent fibrin deposition. Fibrin can be observed in severe AMR in PTCs as detected by IF staining (nephrectomy specimens) (Cornell et al., 2008). This is mainly due to the activation of complement mechanisms and the resultant neutrophil and macrophage chemotaxis and activation. Concerning the Banff grading system (2007), this study found that two out of the nine cases diagnosed as AHR GII would be reclassified as AHR GIII because of the IF detection of fibrin in the wall of small arterioles. This indicates the importance of fibrin staining in graft impairment cases for proper grading. Fibrin detection in peritubular capillaries indicates a more aggressive form of AHR as in the study carried out by (Truong et al., 2007), which correlated the grading with the specific line of treatment. Regarding the relation between the detection of AHR cases using IF C4d and the number of subsequent rejection episodes, in this study, four (18%) out of 22 cases of AHR showed single rejection episodes, whereas the remaining 18 (82%) showed multiple rejection episodes, and in ACR, five (28%) out of 18 cases showed single rejection episodes, whereas the remaining 14 (72%) showed multiple rejection episodes. These results were significant (P value=0.03), indicating increased multiple rejection episodes in AHR than in ACR and confirmed the study carried out by (Poduval et al., 2005). This may be due to the existence of causative antigens (whether major or minor HLA antigens) in the graft with the subsequent triggering of immune mechanisms several times, especially for the AHR type. Regarding the comparison of C4d staining methods, sensitivity and increased interobserver variability have been reported for routinely carried out IHC methods compared with the IF method. In this study, of the 22 (41.5%) cases that were diffusely positive by IF, 10 (18.9%) were moderately stained by IHC (P value=0.01) as in the study carried out by Seemayer et al. (2007) and Colvin and Nickeleit (2006), who demonstrated that IHC staining is less sensitive and may be unreliable; thus, the criteria for positivity are less stringent and oncerning pitfalls in Interpretation:
- False-negative C4d staining is found in areas of necrosis, and care must be taken in some cases to find areas of viable tissue, most easily done in fixed sections.
- The medulla is adequate for interpretation, because medullary capillaries are also targets of C4d. The tubules of the medulla may be misinterpreted as vascular capillaries, especially in frozen tissue.
Arteries and arterioles commonly have C4d, even in kidneys affected by vascular disease. The reason is unknown, perhaps because of autoantibody or no antibody complement activation (Colvin, 2007). In this study, 59% of the entire diagnosed C4d-positive cases responded to AHR treatment, whereas 41% needed ACR treatment as in the study carried out by (Dragun and Rudolph, 2007). This led to the possibility that those cases could be targets for both AHR and ACR. Also, 16.7% of the C4d-negative cases responded to AHR treatment. This negativity for C4d may be attributed to the severe endothelial injury and shedding of the endothelial cells with the bound C4d (Bickerstaff et al., 2008). Thus, from these data, we conclude that C4d plays a pivotal role in the detection and diagnosis of AHR cases with the subsequent proper classification of graft rejection cases according to the Banff schema (2007). It is also very obvious that C4d staining for graft rejection cases plays a major role in modifying the protocol of treatment for better graft survival. Fibrinogen detection in vascular lesions using the IF technique is important as an essential step for the detection of missed cases of fibrinoid necrosis that are specific for AHR cases and were not confirmed by routine light microscopic staining. It also aided the precise reclassification of AHR cases for appropriate treatment.
Conflicts of interest
There are no conflicts of interest.
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