C4d in Acute Rejection After Liver Transplantation—A Valuable Tool in Differential Diagnosis to Hepatitis C Recurrence



Hepatitis C is the most common indication for liver transplantation. Recurrence of HCV is universal leading to graft failure in up to 40% of all patients. The differentiation between acute rejection and recurrent hepatitis C is crucial as rejection treatments are likely to aggravate HCV recurrence. Histological examination of liver biopsy remains the gold standard for diagnosis of acute rejection but has failed in the past to distinguish between acute rejection and recurrent hepatitis C. We have recently reported that C4d as a marker of the activated complement cascade is detectable in hepatic specimen in acute rejection after liver transplantation. In this study, we investigate whether C4d may serve as a specific marker for differential diagnosis in hepatitis C reinfection cases. Immunohistochemical analysis of 97 patients was performed. A total of 67.7% of patients with acute cellular rejection displayed C4d-positive staining in liver biopsy whereas 11.8% of patients with hepatitis C reinfection tested positive for C4d. In the control group, 6.9% showed C4d positivity. For the first time we were able to clearly demonstrate that humoral components, represented by C4d deposition, play a role in acute cellular rejection after LTX. Consequently C4d may be helpful to distinguish between acute rejection and reinfection after LTX for HCV.


liver transplantation


hepatitis C


end-product of activated complement cascade component C4




kidney transplantation


human leukocyte antigen


hepatitis B


hepatitis D


primary biliary cirrhosis


primary sclerosing cholangitis


non-A non-B hepatitis


liver disease






Liver transplantation today is a well-established procedure for curative treatment of various liver diseases. In spite of continuously improving immunosuppressive protocols, acute rejection remains a rather frequent (25–40%) and potentially hazardous complication that is mostly treated by steroid-pulse therapy and/or increased doses of calcineurin inhibitors as well as the introduction of alternative immunosuppressive substances such as MMF or sirolimus (1–3).

Hepatitis C-induced liver cirrhosis is one of the most frequent pathologies requiring liver transplantation (4–6), associated with the high risk of post-transplantation HCV reinfection and consecutive development of liver fibrosis and cirrhosis.

Both acute rejection and hepatitis C reinfection often display the same clinical picture with rising serum transaminases, elevated bilirubin levels and deterioration of productive liver function in the absence of perfusion deficits.

Liver biopsy represents the gold standard for diagnosis of both acute rejection and HCV reinfection, nevertheless, discrimination can be highly difficult due to quite similar display of alterations in the liver specimen (7–9).

False treatment of suspected acute rejection with high-dose pulse steroid therapy in HCV-positive patients can have deleterious effects as hepatitis C virus activity may be severely increased by steroid treatment. Many authors advocate a very restricted use of steroid-pulse treatment even in cases of validated rejection in HCV-positive patients in order to avoid activation of virus replication (8,10–12).

Therefore a specific marker expressed only in rejection but not in HCV reinfection cases would be a great asset to differential diagnosis for HCV-positive patients with clinically suspicious symptoms in order to validate rejection diagnosis.

C4d is an end-product of the activated classical complement cascade, typically detectable in infectious situations and autoimmune disorders stimulating the complement system.

In recent years C4d has become a highly valued tool for diagnosis of acute rejection following kidney transplantation where humoral mechanisms play a by far greater role than after liver transplantation (13–16). Early humoral rejection with consecutive graft loss remains a rather exotic complication following liver transplantation where T-cell-mediated mechanisms are mainly held responsible for organ damage. Only incidental reports of acute humoral rejection, often associated with fatal outcome, are described (17).

In this study we investigated whether profound humoral mechanisms not leading to hyper-acute dramatic rejection do play a role in mainly T-cell-mediated rejection episodes after liver transplantation. Furthermore, we evaluated the potential role of C4d as a valid parameter in differential diagnosis between acute rejection and hepatitis C reinfection in HCV-positive patients (18).

Patients, Material and Methods


We performed retrospective analysis of 97 liver biopsies from liver transplanted patients who had either experienced acute rejection, hepatitis C reinfection or displayed no pathological alterations serving as controls. We investigated 97 patients who had received liver transplantation at our institution between 2000 and 2004. The underlying diseases requiring LTX were hepatitis C-induced cirrhosis, hepatitis B cirrhosis, alcohol-induced cirrhosis, primary biliary cirrhosis, primary sclerosing cholangitis, Morbus Wilson, Budd Chiari Syndrome, polycystic liver disease, alpha-1 anti-trypsin deficiency, autoimmune hepatitis, hepatic Morbus Osler, hemangioendothelioma and cryptogenic cirrhosis.

Patients were divided into three groups:

Group 1 comprised 34 patients with acute rejection (see Table 1). Thirty-two patients had displayed the clinical picture of acute rejection confirmed by consecutive liver biopsy. All these individuals were treated with 500 mg of methylprednisolone per day over 3 to 5 days immediately after histological confirmation of the diagnosis. In 30 of these patients, rejection resolved following steroid treatment with additional dose increase of calcineurin inhibitors.

Table 1. Group1: Acute rejection
PatientsRejection gradeAge (y)SexPost-LTXDiseaseC4d
1I44m0.2 monthsAlcohol
3I62f0.2 monthsHCC+
4II41f24 daysPBC
5II41m7 daysAlcohol+
6II28f9 daysAutoimmune+
7II44f7 daysAutoimmune+
9II18m7 daysAcute LF+
10II48m3.2 monthsHCV, alcohol+
11I59f24 daysPolycystic LD+
12II50f4.2 monthsPBC+
13III50f4 monthsAlcohol
14II27m7 daysHBV
15II55m0.2 monthsHCV
16I57m15 daysAlcohol
17I58m2 monthsAlcohol+
18I49f9 daysCryptogenic
19II30m9 daysPSC
20II49m5 daysHCV
21II67f4 weeksHCV
22II60m6 weeksAlcohol+
24II45f4 weeksCryptogenic+
25I47m3.3 yearsHCC+
26I38f2 weeksAlcohol+
27I60m8 weeksAlcohol+
28I47f3.8 yearsAutoimmune+
29II38f1.8 yearsM.Wilson+
30I18m3 yearsPSC
31III39f7 monthsCryptogenic+
32III51m2 monthsHCV+
33Not scored44f1 yearsHCV+
34Not scored43f3 yearsHCV+

Two patients required OKT 3 treatment leading to successful organ salvage. For two additional patients initially treated for hepatitis C reinfection, the further clinical course clearly demonstrated misled primary diagnosis and revealed rejection in control biopsy.

Group 2 comprised 34 patients (see Table 2). Thirty patients with histologically confirmed hepatitis C recurrence in liver specimen and increased liver enzymes. Four additional patients presented with rising liver enzymes and the suspicion of acute rejection in the liver biopsy and were treated with steroids. However, the further course of the patients who did not respond to rejection therapy proved the occurrence of severe early recurrent hepatitis C.

Table 2. Group 2: Hepatitis-C recurrence
PatientsAge (y)SexPost-LTXDiseaseInterferon before biopsyInterferon after biopsy (≤3 months)Interferon laterC4d
164m8 weeksHCVNoYes 
248m30 monthsHCVYesYes +
344f3 monthsHCV, alcoholNoYes 
437m36 monthsHCV, HBVYesYes +
559m6 monthsHCVNoYes 
652m12 monthsHCVNoNoNo
754m36 monthsHCVYesYes 
845f12 monthsHCVNoYes 
959m12.3 monthsHCVYesYes +
1054f12.1 monthsHCVNoNoYes
1162m36 monthsHCVNoNoNo
1260f36 monthsHCV, HCCYesYes 
1364m36 monthsHCVNoNoYes
1450f36 monthsHCVNoNoNo+
1541f6 monthsHCV,HBVNoNoNo
1643m12 monthsHCV, HCCYesYes 
1765m36 monthsHCV, HCCNoNoNo
1849m36 monthsHCVYesYes 
1967f36 monthsHCVNoNoNo
2049m25 monthsHCVYesYes 
2142m35 monthsHCVNoNoYes
2268m36 monthsHCVNoNoNo
2360f36 monthsHCVNoNoNo
2450f12 monthsHCVNoNoNo
2562f36 monthsHCVNoNoYes
2648m36 monthsHCVNoYes 
2749m12 monthsHCVYesYes 
2845m7 monthsHCVNoYes 
2963m36 monthsHCVNoNoYes
3061m32 monthsHCVYesYes 
3149m30 monthsHCVNoYes 
3249f10 daysHCVNoYes 
3348m8 monthsHCVYesYes 
3445m11 daysHCVNoYes 

Interferon and ribavirin treatment was initiated in 6 patients immediately after biopsy-proven HCV reinfection, 10 patients had already been on interferon and ribavirin, for 18 patients treatment was postponed or not initiated at all due to earlier unresponsiveness or incompliance.

Group 3 comprised 29 patients (see Table 3) with various underlying diseases other than hepatitis C leading to LTX, who had received protocol biopsies 1, 3 or 5 years after liver transplantation. All these individuals displayed no clinical or histological indications for acute or chronic rejection and served as controls in comparison to groups 1 and 2.

Table 3. Group 3: Controls
PatientsAge (y)SexPost-LTXDiseaseC4d
155m12 monthsHBV
253f12 monthsAlcohol
360m36 monthsCryptogenic
465f36 monthsCryptogenic
537f12 monthsM. Rendu-Osler
620m12 monthsAutoimmune
742m12 monthsHBV, HDV
858f12 monthsHaemangioendothelioma
958m12 monthsCryptogenic
1056f12 monthsKlatskin tumor
1160f12 monthsAlcohol, HCC
1241f36 monthsAlcohol
1352m36 monthsAlcohol
1451m12 monthsCryptogenic
1562m12 monthsAlcohol
1652m12 monthsA1-antitrypsine defic.
1758m12 monthsCryptogenic+
1856f12 monthsPBC
1959m12 monthsAlcohol
2063m12 monthsAlcohol, HCC
2148f12Cystic LD+
2249f12 monthsAcute LF
2342m28 daysAlcohol
2447m12 monthsHBV
2545f12 monthsPSC
2655m12 monthsHBV
2763m12 monthsHBV
2864m12 monthsAlcohol, HCC
2960m12 monthsAlcohol, HCC

No episode of acute rejection was present during the 6 months prior to biopsy for all these patients to avoid potential interference with persistent C4d deposits from earlier rejection episodes.


Primary immunosuppression was based on calcineurin inhibitors and steroids in all cases.

Thirteen of 97 patients included in the study received cyclosporine; the other 84 were treated with tacrolimus. Initially, all patients received methylprednisolone, which was reduced or even entirely excluded in the further course. For HCV patients early steroid reduction and complete withdrawal within 2 months after LTX was achieved. In 43 cases, MMF was introduced in doses of 500–2000 mg per day at some point during post-transplant monitoring. For 4 patients sirolimus was administered at 2 mg daily.


Every specimen was taken due to the clinical suspicion of acute rejection, hepatitis C reinfection or as protocol biopsy 6 months, 1, 3 or 5 years after LTX.

Conventional H/E staining was performed for all biopsies, specimens with conventional diagnosis of acute rejection were graded according to the Banff classification of acute cellular rejection (18,19).

Immunohistochemical staining (C4d)

Tissue samples were subjected to C4d specific immune-histological staining and evaluated by two independent pathologists.

After patient selection tissue sections of 1–3 μm were deparaffinized, epitopes were demasked (pressure cooker, citrate buffer, pH 6). Staining was performed using commercially available rabbit polyclonal antibody against C4d (Biomedica, Austria), which was appropriately diluted (1:10).

Antibody binding was detected by using the labeled streptavidin-biotin method (LSAB-Kit+, DAKO, Denmark): Peroxidase-linked streptavidin binds to biotin, which again is linked to the secondary antibody. DAB (3,3 diaminobenzidine; DAKO, Denmark) served as a chromogen for the reaction with peroxidase. Endogenous peroxidase was blocked by H2O2. In a final step nuclear counter-staining with hematoxyline according to Mayer was performed. Successful IH was controlled using tonsil tissue treated as described above; negative controls were obtained by omitting the primary antibody (Figures 2 and 3).

Figure 2.

Immunohistochemistry: C4d deposits along portal endothelium in acute rejection after liver transplantation (200x).

Figure 3.

Immunohistochemistry: C4d-positive staining along endothelial cells of a portal vein, a small artery and portal capillaries in acute rejection after liver transplantation (400x).


Since immunofluorescence is the gold standard in the diagnosis of renal allograft rejection, it was used to validate the results obtained by IH staining.

Therefore, C4d immunofluorescence staining was performed on biopsy specimens showing C4d positivity in IH single staining.

After deparaffinizing the sections (1–3 μm), the epitopes were demasked (citrate buffer, pH 6) and unspecific binding sites were blocked. The primary antibody was diluted (1:10) and added. Avoiding light exposure, slides were incubated with a Cy2-conjugated donkey anti-rabbit secondary antibody (1:100, Dianova, Germany). Finally, nuclear counter-staining was performed. As the fluorescent marker Cy2 emits a green signal, positive C4d staining resulted in green fluorescence (Figure 4).

Figure 4.

Immunofluorescence: capillary C4d deposits in periportal tract in acute rejection after liver transplantation.

Histopathological evaluation

Histopathological evaluation of acute rejection was performed by two independent pathologists according to the Banff schema for grading of liver allograft rejection (Demetris 1997). Acute rejection consequently was qualified as mild, moderate or severe (Table1).

Immunhistochemical stainings were also graded by two independent pathologists without knowledge of the patients' clinical course. Specimens were prepared and paraffinized by a lab technician and then labeled with a code number before forwarding them to the respective pathologist. C4d staining evaluation and diagnosis of H/E-stained specimen were done separately to avoid influence of H/E-diagnosis on evaluation of C4d stained specimen. Both pathologists were blinded concerning the conventional diagnosis in H/E staining as well as to the underlying disease of the patient.


Statistical analysis was performed using SPSS for windows 10.0 (SPSS Inc. Chicago, IL). For comparison of C4d frequency in respective groups, data were subjected to Fisher's exact test for simple cross-tables. Likelihood ratios were calculated by chi-square test for higher dimensional tables. Significance was accepted when p < 0.05.

Sensitivity and specificity as well as confidence intervals and predictive values were calculated by ROC curve analysis using MedCalc statistical software (MedCalc Software, Mariakerke, Belgium).


Twenty-three of 34 patients who by conventional histological examination had been diagnosed with acute cellular rejection displayed C4d-positive staining in their liver biopsy (67.7%) whereas 4 of 34 patients with hepatitis C reinfection were tested positive for C4d (11.8%). In the control group 2 of 29 specimens showed C4d positivity (6.9%).

C4d expression was significantly increased in acute rejection compared to hepatitis C recurrence (p < 0.001) and controls (p < 0.001).

In C4d, positive samples were found along endothelial cells of portal veins, portal arteries and portal capillaries. No distinct distribution pattern of C4d regarding portal vascular structures could be observed. Additionally, no C4d deposits were detected along hepatic veins or sinusoids. Furthermore no difference concerning the distribution of C4d was found between acute rejection and C4d-positive HCV recurrence cases.

Group 1: Nine patients in the rejection group had received LTX for HCV-induced cirrhosis; therefore C4d detection in these cases was especially interesting in order to discriminate between true rejection and possible HCV reinfection. At the time of biopsy, HCV reinfection was apparent in none of the 9 patients, C4d staining was positive in 6 of these 9 patients.

Two of the 6 HCV patients with positive retrospective C4d detection had biopsies 11 days after LTX, a high-risk period for acute rejection that could be confirmed by C4d positivity now. One other patient experienced rejection 3 months after transplantation, a time point when HCV reinfection also is likely to occur. C4d positivity was consistent with the clinical course after steroid-pulse treatment proving rejection. Hepatitis C reinfection in this case could be proved only as late as 27 months after LTX. The fourth patient experienced steroid-resistant rejection 2 months after LTX requiring OKT 3 therapy. Patient numbers 5 and 6 developed hepatitis C reinfection only years later. In summary, the further clinical course proved rejection diagnosis and associated C4d positivity to be correct in these six cases.

The remaining 3 of the 9 HCV patients in the rejection group also proved to have undergone acute rejection as response to anti-rejection therapy and evaluation of the further clinical course revealed. However, C4d staining was negative in these cases, well in line with the overall percentage of C4d positivity among all rejection cases (66.6% and 67.7%).

No correlation between presence and intensity of C4d and severity grade of rejection could be observed (18,19).

Group 2: In the HCV recurrence group 4 of 34 patients (11.8%) presented with C4d-positive liver biopsies. Of these four cases three had received interferon therapy prior to biopsy due to previously proven recurrence of hepatitis C.

Group 3: In the control group, 2 of 29 patients (6.9%) had C4d-positive biopsies, one of them having been transplanted for cryptogenic cirrhosis 2 years before biopsy and never having experienced rejection. However, 3 years later, portal fibrosis and chronic rejection were detected without exact classification of origin. The second patient had received LTX for polycystic liver disease and now displayed a C4d-positive result in the 1 year protocol biopsy showing a metabolic toxic lesion of the parenchyma yet had never experienced rejection episodes (Figure 1).

Figure 1.

Percentage of C4d-positive staining in rejection cases, hepatitis-C recurrence and controls.

The significance for C4d expression as a positive marker of acute rejection displayed a specificity of 90.5% with a sensitivity of 67.7%. The positive predictive value of C4d accurately labeling acute rejection was 75.128% and the negative predictive value was 86.807%. These data were calculated with an area under the curve value of 0.791 (ROC curve, 95% confidence interval 0.696 to 0.867) and a standard deviation of 0.051.


In this study we were able to show for the first time that reasonable amounts of C4d are present in liver tissue in cases of acute rejection following liver transplantation. Significant differences could be detected compared to C4d presence in control biopsies and hepatitis C reinfection patients. These data suggest the activation and involvement of B-cell-related mechanisms in acute rejection episodes after LTX to a far greater extent than previously expected. In previous studies, we were able to show increased presence of complement factors as well as macrophages and plasma cells in liver tissue undergoing acute rejection (20,21). However, no increase of other proliferating cells could be found suggesting migration of plasma cells and macrophages to the spot of immunological activation. Additionally, up-regulation of IgVH genes in liver allograft rejection specimen has demonstrated selected accumulation of B-cells and plasma cells (22).

C4d as the activated component of C4, a central factor of the classical complement pathway, has been established as an indispensable marker tool for acute as well as chronic rejection in kidney transplant patients (15,23,24).

Since its first description by Feucht et al. (25,26) C4d has proven to be a reliable indicator of rejection mechanisms present in the transplanted kidney tissue (16,27–30). After activation of the complement component C4, its degradation product C4d covalently binds to protein and carbon structures directly in the area where it is expressed enabling us to detect it in tissue biopsies. In NTX cases with de novo donor-specific antibodies C4d is expressed in 95% and humoral rejection accounts for as much as 25% of all rejection episodes (15,21).

Humoral mechanisms have been known to play a far greater role in acute rejection following kidney transplantation than in liver transplantation. Steroid-resistant rejection episodes are often of B-cell-related origin and susceptible to plasmapheresis, IVIG or anti-CD 20 antibody treatment (13).

The liver has widely been accepted as the immunologically more “tolerant” organ where rejection mechanisms are almost always based on T-cell-mediated immune response (13). Cases of pure humoral rejection are extremely rare, most often of fulminant nature and reports rather anecdotal (17,22). However, Sawada et al. demonstrated humoral immunity to be partially responsible for allograft damage in acute rejection after LTX (31).

It was our aim to evaluate whether C4d can be detected in liver biopsy specimen of patients with acute rejection (AR) episodes and may, therefore, serve as a specific marker supporting diagnosis of AR. Our special interest was dedicated to the differences in C4d expression compared to hepatitis C reinfection patients after LTX. Due to the immunologic processes within the transplanted organ following HCV recurrence differential diagnosis between acute rejection and HCV reinfection can be of great difficulty to the pathologist, especially in cases of mild rejection and de novo HCV reinfection (7,8). Mononuclear infiltrates of the portal tract are widely present in both cases challenging accurate differential diagnosis (32–34). In the past, this phenomenon has led to false histological diagnosis of acute rejection in HCV reinfection cases with consecutive steroid-pulse therapy and fatal effects on virus activation (4,10).

Many authors advocate steroid-free rejection therapy regimes for hepatitis C patients in order to minimize viral stimulation as steroid application for presumed rejection has been associated with significantly impaired outcome for HCV patients (8,35).

Several potential markers have been investigated to improve differential diagnostic criteria (36–39) yet no clinically applicable routine could be established. Ciccorossi et al. demonstrated anti-HCV IgM to be a highly specific marker of HCV recurrence when measured quantitatively. However, continuous HCV serological data need to be available in order to set quantitative levels into the right context. HCV-RNA was found to be expressed to a greater extent in patients transplanted for hepatitis C if HCV reinfection was present than in acute rejection yet no decisive quantitative value can be determined (39).

Srekunar et al. were able to detect a significantly greater expression of MHC class I and II associated genes in liver specimen in case of acute rejection than in HCV recurrence.

Unfortunately, genetic characterization is a complicated and time-consuming procedure that cannot serve for immediately required diagnosis in the clinical situation (36).

C4d staining is a fast and widely available procedure well established in the histological evaluation of kidney transplant rejection today. Hence liver biopsy specimen can be immediately stained for C4d either in cryopreserved or paraffinized tissue granting C4d-associated evaluation within hours (20).

We therefore looked at C4d as a potential marker for diagnostic discrimination between acute rejection and HCV reinfection in patients who had received LTX for HCV-induced cirrhosis.

Our results clearly show great differences in C4d expression between the respective groups. We were able to demonstrate a specificity of 90% for C4d positivity indicating acute rejection. However, sensitivity was only 67.7%, therefore we advocate the application of C4d as a supportive yet not secure marker of acute rejection after LTX. These numbers are well in line with expression rates after kidney transplantation where detection of C4d in rejection cases varies between 50% and 60% (12,26,27). As discussed above, retrospective scrutinization of individual cases has led us to suspect misled diagnosis due to insufficient conventional biopsy in specific cases.

Analysis of the 97 cases included in this study revealed false diagnosis by conventional histological staining for at least 6 patients; 4 individuals with hepatitis C reinfection who were misdiagnosed as undergoing acute rejection and 2 HCV patients who did not experience hepatitis C recurrence but acute rejection instead. Availability of C4D staining at the time of diagnosis might have spared these patients from false diagnosis followed by inadequate and potentially hazardous treatment.

Among the 4 C4d-positive HCV patients 3 had received interferon treatment prior to biopsy possibly inducing humoral mechanisms including complement activation in hepatitis C-positive patients. Baid et al. could show a remarkable increase in C4d-positive rejection episodes in kidney transplant patients suffering from chronic hepatitis C infection, which is possibly due to enhanced cell surface expression of HLA alloantigens (40). However, the question whether interferon treatment may induce susceptibility to acute or chronic rejection in solid organ transplantation is being discussed controversially. Cosimi et al. have shown interferon not to increase frequency of acute rejection after LTX (41) whereas many other authors advocate an increased risk for rejection episodes under interferon treatment (42,43).

Jain et al. performed a large retrospective study analyzing 105 LTX patients with hepatitis B and C who had received interferon-alpha treatment and could not detect a significant increase in rejection rates. However, patients in this study had received slightly higher steroid doses than control patients without interferon treatment (44).

In conclusion, we state that C4d as an established marker for acute rejection in kidney transplantation also plays a considerable role in acute rejection following liver transplantation suggesting the involvement of humoral mechanisms to a greater extent than currently accepted.

For the first time we were able to detect significantly stronger C4d deposits in liver biopsies of patients suffering from acute rejection than in rejection-free individuals.

Especially in differential diagnosis to HCV recurrence, which in conventional histological staining can be rather demanding, C4d may be able to contribute to more accuracy in specifying exact diagnosis in the future.