- Top of page
- Material and Methods
In renal transplant patients with de novo donor-specific antibodies (dnDSA) we studied the value of microcirculation inflammation (MI; defined by the addition of glomerulitis (g) and peritubular capillaritis (ptc) scores) to assess long-term graft survival in a retrospective cohort study. Out of all transplant patients with standard immunological risk (n = 638), 79 (12.4%) developed dnDSA and 58/79 (73%) had an indication biopsy at or after dnDSA development. Based on the MI score on that indication biopsy patients were categorized, MI0 (n = 26), MI1 + 2 (n = 21) and MI ≥ 3 (n = 11). The MI groups did not differ significantly pretransplantation, whereas posttransplantation higher MI scores developed more anti-HLA class I + II DSA (p = 0.011), showed more TCMR (p < 0.001) and showed a trend to C4d-positive staining (p = 0.059). Four-year graft survival estimates from time of indication biopsy were MI0 96.1%, MI1 + 2 76.1% and MI ≥ 3 17.1%; resulting in a 24-fold increased risk of graft failure in the MI ≥ 3 compared to the MI0 group (p = 0.003; 95% CI [3.0–196.0]). When adjusted for C4d, MI ≥ 3 still had a 21-fold increased risk of graft failure (p = 0.005; 95% CI [2.5–180.0]), while C4d positivity on indication biopsy lost significance. In renal transplant patients with de novo DSA, microcirculation inflammation, defined by g + ptc, associates with graft survival.
- Top of page
- Material and Methods
Antibody involvement in renal allograft rejection was first described in the 1960s and, after the discovery of C4d as a biomarker in 1991, criteria for antibody-mediated rejection (AMR) were developed . The Banff consensus for allograft pathology defines AMR as a triad of circulating donor-specific antibodies (DSA), C4d deposition in peritubular capillaries and specific histological parameters. Both acute and chronic AMR are associated with inferior graft outcome [2-4].
The diagnostic criteria for AMR are currently undergoing review , with the increasing recognition of C4d-negative AMR based on transcript analysis studies  and on observations in sensitized patients with preformed DSA . In these patients, microcirculation inflammation (MI), defined by the combination of glomerulitis (g) and peritubular capillaritis (ptc) scores, is associated with worse outcome . In the setting of de novo DSA (dnDSA), MI has also been noted . On a population scale, de novo antibodies targeted at the transplanted donor antigens are associated with worse outcome  but not all patients with dnDSA develop detrimental damage to the graft [10, 11].
Our aim was to define the incidence of C4d-positive AMR and C4d-negative AMR in the first indication biopsy up to 1 month before or anytime after dnDSA developed in a group of patients with dnDSA, based on microcirculation inflammation (MI) and C4d scores. The value of the MI score was compared to C4d staining to establish a correlation with outcome in renal transplant patients with dnDSA.
- Top of page
- Material and Methods
All renal transplant recipients with standard immunological risk between 2005 and 2010 (n = 638) were analyzed at least once after transplantation for the formation of anti-HLA antibodies by Luminex. Seventy-nine (12.4%) patients were found to have dnDSA at a median time of 3.8 [IQR 0.6–10.8] months after transplantation; with 24 patients displaying only anti-HLA class I DSA, 33 only anti-HLA class II DSA and 22 patients having both. Of these 79 patients, 58 (73.4%) had an index biopsy. These 58 patients received a total of 255 biopsies, reaching an average of 4.4 biopsies per patient.
The patient demographics are described in Table 1, comparing the total renal transplant population minus patients with dnDSA (n = 559), to the full group with dnDSA (n = 79) and to the group with dnDSA and an index biopsy (n = 58). Graft survival at 4 years after transplantation was estimated for the four groups: total standard immunological risk population 88.2% (n = 638), total population minus patients with dnDSA 90.6% (n = 559), patients with dnDSA 73.2% (n = 79) and patients with dnDSA and with index biopsy 70.7% (n = 58) (Figure 1), showing reduced graft survival estimates in the recipients with dnDSA. There were no significant differences between the estimated graft survival rates of all dnDSA patients and those 58 patients with a biopsy. Further results described below focus on the 58 dnDSA patients with an index biopsy.
Table 1. Clinical demographics comparing all transplant recipients with standard immunological risk to those that developed de novo DSA
| ||All||De novo DSA||De novo DSA with|
| ||n = 559||n = 79||biopsy n = 58|
|Age (yr; median [IQR])||50 [39–59]||45 [34–55]||45 [34–55]|
|Gender (% male)||68.2||64.6||65.5|
|Ethnicity (% cauc/asian/afrocar/other)||48/33/14/5||38/43/14/5||40/43/14/3|
|Total HLA mismatches||3.1 (±1.6)||3.6 (±1.3)||3.9 (±1.3)|
|HLA-class 1 MM (mean ±SD)||2.3 (±1.2)||2.4 (±1.2)||2.8 (±1.0)|
|HLA-class 2 MM (mean ±SD)||0.9 (±0.7)||1.2 (±0.6)||1.1 (±0.6)|
|Induction therapy (% Campath)||79.6||91.1||89.7|
|FU graft survival (mnth; median [IQR])||32 [20–50]||33 [19–50]||33 [20–50]|
|FU patient survival (mnth; median [IQR])||34 [22–51]||39 [25–53]||40 [25–53]|
Figure 1. Overall death-censored graft survival stratified by total standard immunological risk population (n = 638), total population minus patients with dnDSA (n = 559), all de novo DSA patients (n = 79) and de novo DSA patients with an indication biopsy from 1 month before or after dnDSA developed (n = 58).
Download figure to PowerPoint
The dnDSA patients (n = 58) were divided into three categories based on MI scores assessed on the index biopsy; MI0, MI1 + 2 and MI ≥ 3. Eighteen (31%) recipients had a positive g score; 31 (53%) had a positive ptc score; 17 (29%) biopsies showed both g and ptc positivity. An MI score of 1 was mostly represented by a solitary ptc1 score (91%). In Table 2 the three groups were compared and analyses showed significant differences between the three groups for posttransplantation factors; development of both donor-specific HLA antibodies class I and II (p = 0.011), TCMR on index biopsy (p>0.001) and a trend for C4d staining (p = 0.059).
Table 2. Comparison of microcirculation inflammation score groups for demographics, immunological risk factors and rejection characteristics
| ||MI 0||MI 1 + 2||MI ≥3|| |
| ||n = 26||n = 21||n = 11||p-Value|
|Age (yr; mean ± SD)||44 [38–57]||51 [42–56]||54 [47–68]||0.22|
|Gender (% male)||67||50||25||0.13|
|Age (yr; mean ± SD)||46 [33–54]||44 [35–56]||42 [32–60]||0.98|
|Gender (% male)||62||57||91||0.14|
|Ethnicity (%) cauc/asian/afrocar/other||31/50/19/0||38/43/14/5||64/27/0/9||0.27|
|Total HLA mismatches||4.0 (±1.0)||3.7 (±1.5)||4.0 (±1.3)||0.61|
|HLA-class I MM (mean ± SD)||2.8 (±1.0)||2.7 (±1.0)||2.8 (±1.0)||0.90|
|HLA-class II MM (mean ± SD)||1.2 (±0.6)||1.0 (±0.7)||1.2 (±1.2)||0.72|
|Retransplantation (% yes)||8||10||27||0.24|
|Preemptive (% yes)||8||14||9||0.85|
|Donor type (% DDRT)||62||33||64||0.12|
|Induction therapy (% Campath)||96||86||82||0.27|
|HLA-antibodies (non-DSA) (% yes)||35||19||18||0.45|
|Time to DSA (mnth; median [IQR])||2.9 [0.5–6.3]||4.7 [0.6–11]||5.2 [3.7–7.7]||0.46|
|Time to Bx (mnth; median [IQR])||4.1 [0.8–14]||6.0 [0.6–19]||8.8 [5.6–27]||0.65|
|Total follow-up (mnth; median [IQR])||31 [20–50]||45 [34–52]||48 [28–52]||0.18|
|Anti-HLA class I&II DSA (% yes)||31||19||73||0.011|
|Anti-HLA class I DSA (% yes)||69||43||91||0.20|
|Anti-HLA class II DSA (% yes)||62||76||82||0.42|
|MFI >1000 (% yes)||62||62||91||0.18|
|TCMR (%≥type 1A)||0||33||73||<0.001|
|C4d staining (% focal/diffuse)||23||29||64||0.059|
Graft loss occurred in 13/58 (22%) patients in this cohort, and was attributed to AMR in 5 patients, whereas in 7 patients AMR was present but in combination with other factors, and 1 was not related to AMR (Table 3). Patient survival did not differ between the three MI groups with 4-year patient survival estimates of MI0 91%, MI 1 + 2 91% and MI ≥ 3 100% (p = 0.468; data not shown [DNS]).
Table 3. All 13 graft failures within the cohort of 58 de novo DSA patients with index biopsy
| || || || || || ||On subsequent biopsies|| || |
| || || || || ||At index biopsy|| || || |
| || ||Mnth||HLA|| || ||Max||Max||Max|| ||Mnth|| |
| ||MI||DSA||class||Mnth||C4d||MI||cg||C4d||MI||cg||Mnth||graft|| |
| ||group||positive||DSA||biopsy||(0–3)||(0–6)||(0–3)||(0–3)||(0–6)||(0–3)||TG||failure|| |
|1||MI0||0.3||1 + 2||0.7||0||0||0||3||1||0||–||2.9||Ureteric complications|
|2||MI1 + 2||3.3||1 + 2||3.7||0||2||0||3||5||3||14.1||17.1||acute AMR + TG + renal artery stenosis|
|3||MI1 + 2||2.9||1 + 2||7.5||1||1||0||1||2||0||–||39.6||Pyelonephritis + TMA|
|4||MI1 + 2||17.7||1 + 2||17.5||0||1||0||0||2||0||–||23.7||C4d-negative acute AMR + TCMR + pyelonephritis|
|5||MI1 + 2||5.7||2||5.3||3||1||0||3||2||0||–||12.9||Acute AMR (reduced IS) + OI|
|6||MI ≥ 3||9.4||1 + 2||8.8||0||4||2||2||5||3||8.8||20.1||TG + TCMR|
|7||MI ≥ 3||36.0||2||37.0||1||5||3||2||5||3||37.0||45.2||acute AMR + TG|
|8||MI ≥ 3||4.0||1 + 2||27.4||3||3||3||3||6||3||27.4||38.4||TG|
|9||MI ≥ 3||5.1||1 + 2||8.6||3||4||1||3||5||3||8.6||17.4||TG|
|10||MI ≥ 3||17.4||1 + 2||25.9||2||4||3||1||5||3||25.9||49.0||TG + TCMR|
|11||MI ≥ 3||0.2||1 + 2||0.2||3||3||0||2||3||0||–||3.0||Acute AMR + pyelonephritis|
|12||MI ≥ 3||5.2||1 + 2||4.9||3||5||0||3||4||3||14.8||35.5||TG + TCMR + pyelonephritis|
|13||MI ≥ 3||6.1||1 + 2||9.1||3||3||0||3||5||3||15.9||18.5||TG + TCMR + recurrent GN|
Microcirculation inflammation is associated with graft survival in patients with de novo DSA
The three MI groups were compared for graft survival from time of index biopsy (Figure 2A) showing a profound decline in graft survival for the patients who had a high MI score (MI ≥ 3) on the index biopsy. The risk of renal graft failure in the group showing an MI ≥ 3 on their index biopsy was 24 times the risk of renal graft failure in the group with an MI0 score (p = 0.003; 95% confidence interval [CI] [3.0–196.0]). Graft survival analysis comparing C4d-positive to C4d-negative staining on the index biopsy (Figure 2B) showed a much less pronounced distinction that did not quite reach statistical significance. Patients with a C4d-positive score had a 2.9 fold increased risk of graft failure (p = 0.066; 95% CI [0.9–9.3]). Albeit both parameters seemed to be powerful risk factors for graft survival, when the MI score was C4d adjusted, patients with an MI ≥ 3 still had 21-fold increased risk of graft failure (p = 0.005; 95% CI [2.5–180.0]), whereas a C4d-positive score lost significance with a 1.4-fold increased risk of allograft failure (p = 0.570; 95% CI [0.4–5.0]). On a separate analysis, the area under the ROC was poor for C4d-positive staining (0.64) whereas it was good for MI score (0.85). Our model showed that MI score is a significantly better predictor of graft loss (p = 0.006, Figure 3). When only diffuse C4d-staining (n = 10) was considered positive, C4d-positivity did associate with a worse survival estimate (p = 0.001; DNS) and in multivariate analysis retained significance with a fourfold increased risk of graft failure, independent of the MI score (p = 0.044; 95% CI [1.04–15.7]). The area under the ROC was again poor (0.69; DNS).
Figure 2. Death-censored graft survival from time of index biopsy comparing (A) the 3 microcirculation inflammation groups, and (B) C4d-positive versus C4d-negative staining. (A) Microcirculation inflammation (MI) is defined by the addition of the glomerulitis and peritubular capillaritis score and separated into three categories based on the MI score on the first indication biopsy around or after de novo DSA. (B) C4d staining of peritubular capillaries scored on the first indication biopsy around or after de novo DSA.
Download figure to PowerPoint
Figure 3. ROC analysis comparing C4d and MI score. Sensitivity (Y-as) and 1-specificity (X-as) are displayed for dichotomized C4d staining (discontinuous line; negative/minimal staining vs. focal/diffuse staining) and categorized MI score (solid line; MI0, MI1 + 2, MI ≥ 3). Reference line is depicted in gray. ROC analysis was performed using a logistic model, defining outcome as graft loss within the follow-up.
Download figure to PowerPoint
Based on findings in the index biopsy, in the MI0 group (n = 26), 24/26 were rejection free, 2 were suspicious for TCMR and 6/26 were C4d-positive (4 focal, 2 diffuse). In the MI1 + 2 group (n = 21), 12/21 were TCMR free, 7 had TCMR (4 grade I, 3 grade II) and 2 were suspicious for TCMR. By definition all 21 patients had MI + DSA so were at least suspicious for AMR, depending on C4d status. Six of the 21 were C4d-positive (acute AMR; 3 focal, 3 diffuse) and 1/6 also had TG; 15/21 were C4d-negative, none with TG. In the MI ≥ 3 group (n = 11), 1/11 was TCMR free, 8 had TCMR (2 grade I, 6 grade II) and 2 were suspicious for TCMR. Seven of the 11 were C4d-positive (acute AMR; 2 focal, 5 diffuse), and 3/7 also had TG. There were 4/11 C4d-negative cases (suspicious for acute AMR), and 3/4 also had TG (Table 3, Figure 4). Total incidence of (C4d-positive) acute AMR was 22.4% (13/58 patients) and total incidence of (C4d-negative) suspicious for AMR was 33% (19/58 patients).
Figure 4. Flowchart depicting attribution of graft failures. Microcirculation inflammation and C4d peritubular capillary staining scores are based on (immuno-) histological parameters in the index biopsy. These scores are shown in relation to the attributed causes for graft failure. Gray boxes indicate C4d+ at the time of index biopsy or on a later biopsy. aAMR = acute antibody-mediated rejection; GN = glomerulonephritis; MI = microcirculation inflammation; OI = opportunistic fungal infection; IS = immunosuppressive drugs; PN = pyelonephritis; RAS = renal artery stenosis; TCMR = T cell mediated rejection; TG = transplant glomerulopathy; TMA = thrombotic microangiopathy.
Download figure to PowerPoint
TG was present in 7/58 (12%) index biopsies, only in the MI positive groups. Including follow-up biopsies, 16/58 patients developed TG; 1 (4%), 6 (29%) and 9 (82%) in the MI0, MI1 + 2 and MI ≥ 3 group, respectively. When graft survival analysis was adjusted for having TG on the index biopsy, patients with an MI ≥ 3 still had a 16-fold increased risk of graft failure compared to patients with an MI0 score (p = 0.014; 95% CI [1.8–149.3]). Patients with TG on the index biopsy had a 9.6 times increased risk of graft failure (p = 0.001; 95% CI [2.7–35.9]) for which significance was lost after multivariate analysis including MI and C4d score.
- Top of page
- Material and Methods
Formation of anti-HLA antibodies after transplantation, and particularly DSA, is associated with acute rejection, chronic rejection and graft loss [10, 15-19]. We set out to study patients with de novo DSA for biopsy features that distinguish those who progress to graft failure from those who retain graft function. We observed that patients with severe microcirculation inflammation (MI ≥ 3) on the first indication biopsy taken within 1 month or after dnDSA occur, are at a significantly increased risk for graft failure. The MI score is better at discriminating graft failure from survival than C4d staining in this cohort of patients with dnDSA. We found that, in the medium term at least, patients without microcirculation inflammation (MI0) on their index biopsy behaved like the standard immunological risk patients who did not develop dnDSA (4-year graft survival rates after transplantation, 91.7% vs. 90.6%, respectively).
In two prospective studies on indication biopsies, late biopsies (>1-year posttransplant) often coincided with de novo anti-HLA class II DSA (with or without anti-HLA class I DSA), MI, microcirculation damage (TG and peritubular capillary basement membrane multilayering) and graft loss [8, 20]. In a study on protocol biopsies of patients with preformed DSA, MI and anti-HLA class II DSA were correlated with bad outcome independent of C4d . In a study of standard immunological risk patients, approximately 15% developed dnDSA late after transplantation predicted by nonadherence and HLA-DRβ1 mismatch . Significantly more ptc was noted in 6-month protocol biopsies in patients with dnDSA compared to those without . In this retrospective study, we find that anti-HLA class II DSA development is associated with worse survival and although there was a trend of later biopsy time with increasing MI score, this was not significant. Our aim was to distinguish within the dnDSA patients those who would progress to graft failure from those who would retain graft function. By using the MI score, shown to correlate with DSA, we found a way to do so.
Our transplant center employs a steroid-sparing mono-immunosuppressive regimen, consisting of alemtuzumab induction with tacrolimus maintenance therapy in the majority of renal transplant recipients with standard immunological risk. The 13% incidence of dnDSA formation in our population is similar to other centers  Time to development of dnDSA is shorter than some reports and similar to others [8, 11, 20-22]. One group, using a steroid-based regimen, describes an incidence of dnDSA in 27% with 90% of protocolized samples showing dnDSA development at the 1 or 6 month posttransplantation sample . Currently a US-based NIH-supported trial is underway (CTOT-02) which has as a first outcome para-meter the screening for incidence and timing of alloantibody development. Future results of this trial might shed light on AMR and DSA incidence in different induction protocols.
The diagnostic criteria for AMR are currently being revised to include C4d-negative cases [5-7, 23, 24]. In a recent study in mostly nonpresensitized renal transplant patients, MI>0 in late biopsies indicated the presence of DSA and predicted inferior graft survival . The cut-off for defining ptc as positive was higher in this study than in ours, making direct comparison difficult. Using the Banff criteria for ptc [13, 14], we found that patients with dnDSA and an MI ≥ 3 did poorly, even if C4d was negative. The results in patients with MI scores of 1 or 2 were less clear cut. We also noted that in patients with dnDSA and MI, C4d staining could fluctuate between positivity and negativity on follow-up biopsies, as noted in presensitized patients by others . In this respect, cumulative C4d scores may prove a more useful prognostic indicator than individual measurements. We also confirm the existence of rare C4d-positive cases (n = 6/26) with stable clinical features and the persistent absence of MI on biopsy (MI = 0), at least in the medium term. We do not know yet whether these cases represent a form of accommodation or of indolent chronic rejection, which only time will tell. MI0 patients had good outcomes, whether C4d was positive or not. These patients are unlikely to be responders to treatment as only 6/26 had received augmented immunosuppression. A diagnostic definition of C4d-negative AMR could be established by defining a threshold for MI with the use of long-term survival data. However, a consensus is necessary on how to define the MI score, before such diagnostic criteria can be agreed upon.
We acknowledge the limitations of this retrospective study. All renal transplant patients at our center had at least one test for DSA, but the timing of DSA sampling was not standardized. There was no established protocol for biopsy timing either, so the timing to detection of DSA and the timing of the index biopsy were biased. Some cases of dnDSA could have been missed as HLA-DP specificities are unknown and no tests were employed for non-HLA DSA such as major histocompatibility complex class I chain-related gene A . The DSA described are however de novo, and we included only indication biopsies.
In patients who develop de novo DSA, the microcirculation inflammation score, defined by glomerulitis plus peritubular capillaritis, is associated with inferior graft survival. Much of this can be attributed to clinical episodes of AMR according to established criteria, but not the C4d-negative cases. Whether treating early dnDSA development or B cell immunity may improve outcome in these patients still needs to be established. Patient-tailored administration of augmented immunosuppression is crucial hence the importance of continued biopsy and DSA screening in standard immunological risk patients. More research is necessary to determine if the MI score as a distinctive parameter for outcome holds in other renal transplant units and in comparison with other clinical and serological features.