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Keywords:

  • Acute humoral rejection;
  • alloantibody;
  • human leukocyte antigen;
  • kidney transplant;
  • positive crossmatch;
  • single antigen beads

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

We examined the course of donor-specific alloantibody (DSA) levels early after transplant and their relationship with acute humoral rejection (AHR) in two groups of positive crossmatch (+XM) kidney transplant recipients: High DSA group—41 recipients with a baseline T- or B-cell flow crossmatch (TFXM, BFXM) channel shift ≥300 (molecules of equivalent soluble fluorochrome units (MESF) of approximately 19 300) who underwent pretransplant plasmapheresis (PP), and Low DSA group—29 recipients with a baseline channel shift <300 who did not undergo PP. The incidence of AHR was 39% (16/41) in the High DSA group and 31% (9/29) in the Low DSA group. Overall, mean DSA levels decreased by day 4 posttransplant and remained low in patients who did not develop AHR. By day 10, DSA levels increased in patients developing AHR with 92% (23/25) of patients with a BFXM >359 (MESF of approximately 34 000) developing AHR. The BFXM and the total DSA measured by single antigen beads correlated well across a wide spectrum suggesting that either could be used for monitoring. We conclude that AHR is associated with the development of High DSA levels posttransplant and protocols aimed at maintaining DSA at lower levels may decrease the incidence of AHR.


Abbreviations: 
AHG

antihuman globulin

AHR

acute humoral rejection

ASC

antibody-secreting cells

ΔCr

change in serum creatinine at the time of biopsy

DSA

donor-specific antibody

FXM

flow crossmatch

HLA

human leukocyte antigen

IVIG

intravenous immunoglobulin

MESF

molecules of equivalent soluble fluorochrome units

MFI

mean fluorescence index

MΔCr

maximum delta creatinine during rejection episode

PC

plasma cell

PP

plasmapheresis

SABs

single antigen beads

+XM

positive crossmatch

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

Donor-specific alloantibody (DSA) is an increasingly common finding in renal transplant candidates (1). With the development of more sensitive assays, it is now clear that DSA levels vary widely among these patients and that the serum level of DSA may be the major determinant of allograft injury (2). For example, patients with high levels of DSA at the time of transplant appear to have a high risk for the development of hyperacute rejection. We have previously reported that 20% of patients who were transplanted with a positive T-cell antihuman globulin (AHG) crossmatch developed hyperacute rejection, while none of the patients who were desensitized to the point of a negative T-cell AHG crossmatch developed hyperacute rejection (2).

Even when hyperacute rejection is avoided, sensitized patients still have a high incidence of acute humoral rejection (AHR) in the first few weeks after transplantation (3). We hypothesize that posttransplant DSA levels correlate with antibody-mediated allograft injury. Thus, the primary aim of the current study was to examine the posttransplant course of DSA levels in positive crossmatch (+XM) kidney transplant recipients as measured by either crossmatch or solid phase assays and to determine their relationship with histologic injury.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

Patients

These studies were carried out with informed consent using protocols approved by the Institutional Review Board of the Mayo Foundation and Clinic and involved a retrospective review of consecutive +XM recipients of ABO compatible kidney transplants at our institution performed between January 1, 2005, and January 10, 2007.

Detection of DSA

Details of the flow cytometric crossmatch, T-cell AHG crossmatch and the SAB (LABScreen™, One Lambda, Canoga Park, CA) assays have been described previously (2,3). When pretransplant plasmapheresis (PP) was performed, the serum DSA determined was from serum collected prior to PP treatment. When thymoglobulin was used, it was extracted from the serum prior to assaying (4). Results of the TFXM and BFXM are reported as channel shifts with a value ≥52 being positive for the TFXM and a value ≥106 being positive for the BFXM (pronase is routinely used in the FXM assay to reduce nonspecific binding sites). In Low DSA recipients, the baseline antibody level is reported as the FXM performed on the day prior to transplant, whereas, in High DSA patients the baseline antibody level refers to the FXM level prior to desensitization. In most instances, the channel shift on the BFXM was greater than the TFXM because the BFXM detects both anti-Class I and anti-Class II antibody and is the primary result that is presented here. However, in rare instances, the TFXM was slightly higher than the BFXM, and clinically we evolved to require both results to be <300 on the day of transplant.

FXM data are presented here as channel shifts using a 1024 linear scale. The channel shift is the observed number of channels (raw fluorescence) subtracted by the number of channels observed from the negative control. However, FXM techniques including the scale may vary widely between laboratories; thus, we retrospectively estimated the molecules of equivalent soluble fluorochrome units (MESF) that correlate with the channel shift in our FXM assays (5).

Conversion to MESF units

Using the same settings and conditions implemented in our FXM, the channel shift of commercially available, standardized MESF beads (Quantum™ FITC MESF Premix, Bangs Laboratories, Fishers IN) was determined as per the manufacturers protocol. A standard curve was generated by plotting fluorescence intensity for five different standardized beads against known MESF units (Quickcal software, Bangs Laboratories). Each set was run on 15 different days. The standardized curve was generated using a linear regression model with added estimation of a within-bead correlation (more formally a mixed model with a compound symmetric correlation structure). The standardized curve and model-based 95% confidence intervals are presented in Figure 1. The conversion formula was determined to be: number of channels (raw or uncorrected) =−528.58 + 104.22*log (MESF). From this equation, the average raw fluorescence of our negative control/unstained B-lymphocytes of 200 channels represents a MESF value of approximately 1100. A channel shift of 300 (raw fluorescence of 500 or 300 channels above the background) is a MESF of approximately 19 300 and a channel shift of 359 is a MESF of approximately 34 000. This range of positive values is similar to that described in another study correlating FXM to MESF in which ‘strongly positive’ crossmatches had MESF values >18 000 and very weak positive crossmatches had MESF values of approximately 2000 (6).

image

Figure 1. Correlation between fluorescence channels and molecules of equivalent soluble fluorescence units (MESF) in our laboratory. This standardized curve was generated retrospectively to allow comparisons between our laboratory's flow crossmatch channel shift data to data available in other laboratories. The curve was generated via a linear regression model with the added estimation of a within-bead correlation. The observed data and means are presented with the model-based 95% CIs. A BFXM channel shift of 300 that is the cutoff between high DSA and low DSA used in these studies represents a total channel number of 500 (the average raw fluorescence of unstained B cells is 200 channels in our laboratory) and a MESF of approximately 19 300. The BFXM channel shift of 359, below which AHR did not occur in this study, corresponds to 559 total channels or a MESF of approximately 34 000.

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Single antigen bead measurements

Results of the SABs are reported as the mean fluorescence index (MFI). This value is calculated by [(raw data value from specific patient bead − raw data value of patient negative control bead) − (raw data count of the same specific bead negative control serum − raw data value of the negative control bead)]. The significance of SAB data was evaluated in three different manners: (1) using the highest single DSA MFI (Class I or Class II); (2) using the highest Class I DSA + highest Class II DSA MFI and (3) adding each of the Class I DSA + each of the Class II DSA MFI to determine a total DSA.

Desensitization protocol

Recipients with a baseline T-cell flow crossmatch (TFXM) or B-cell flow crossmatch (BFXM) channel shift ≥300 (High DSA group) underwent pretransplant desensitization to achieve both T and BFXM channel shifts <300 on the day of transplant and received four to seven posttransplant PP treatments as previously described (3). This value was chosen because all prior cases of hyperacute rejection in our program occurred in patients with a positive T-cell AHG crossmatch at the time of transplant that corresponded to a TFXM channel shift >300. Recipients with a T or BFXM channel shift <300 at baseline (Low DSA group) received no pretransplant PP but were monitored closely with daily serum creatinine and posttransplant DSA levels (TFXM, BFXM and single antigen beads [SABs] performed on postoperative days 4, 10 and 28). All patients received induction with antithymocyte globulin (Thymoglobulin®; 1.5 mg/kg/day × five- to seven doses) and maintenance immunosuppression with tacrolimus, mycophenolate mofetil and prednisone as previously reported (2).

Renal allograft biopsies

Ultrasound-guided percutaneous biopsies were processed for light microscopy and immunofluorescence staining for C4d. All cases of suspected AHR were confirmed by allograft biopsy. Initially, we also performed protocol biopsies on all +XM kidney transplant recipients on days 4 and 7. However, later in the study, 13 patients in the Low DSA group and 3 patients in the High DSA group who had stable posttransplant courses were not biopsied. Thus, it is possible that some cases of ‘subclinical’ humoral rejection were not identified. AHR was diagnosed using standard criteria from the Banff working group (7,8). In addition, biopsies were further scored using a graded scale as follows: no humoral rejection (Figure 2A); C4d deposition only (C4d+ peritubular capillaries by immunofluorescence without evidence of acute histologic changes by light microscopy, Figure 2B); moderate AHR (mild tubular damage, neutrophil infiltration, Figure 2C) and severe AHR (glomerular thrombosis, necrosis, mesangiolysis, Figure 2D). C4d deposition alone in the absence of histologic injury on light microscopy was not considered AHR.

image

Figure 2. Histologic spectrum of acute humoral rejection. (A) Histologically normal biopsy that was negative for C4d on immunofluorescence. (B) Histologically normal biopsy that showed diffuse, bright staining of peritubular capillaries for C4d by immunofluorescence (inset). (C) Acute tubular injury, with dilatation of the tubules and flattening of the tubular epithelium (arrow; moderate rejection); immunofluorescence studies showed diffuse, bright staining of peritubular capillaries for C4d (inset). (D) Focal glomeruli with thrombi within the capillary lumens (arrows; severe rejection); C4d was diffusely positive in peritubular capillaries by immunofluorescence (inset). All histologic sections pictured are stained by hematoxylin and eosin.

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Histologic findings were correlated with DSA levels performed on serum collected within 24 h of the biopsy. If the recipient progressed in rejection severity (based on histologic injury), then each time point was included in the analysis. Once the recipient reached the maximum rejection severity, subsequent biopsies and DSA levels were excluded from further analysis. This protocol was followed to limit the potential effects of treatment (plasmapheresis; IVIG) for AHR on antibody levels. Thus, a total of 84 biopsies (from 53 recipients) were included for analysis.

Serum creatinine

The posttransplant creatinine was reviewed for all recipients. The change in serum creatinine from a posttransplant nadir to the serum level at the time of biopsy (ΔCr) was stratified according to rejection severity. Additionally, the maximum delta creatinine (MΔCr) was determined by subtracting the nadir creatinine after transplant from the peak creatinine during the 28-day study period for patients who did not have evidence of AHR. For those patients who did experience AHR, the MΔCr was calculated by subtracting the nadir creatinine after transplant from the maximum creatinine level during the rejection episode. The MΔCr was also stratified according to rejection severity.

Statistical analysis

Paired t-tests were used to compare mean antibody levels across time points (pretransplant, day 4, day 10 and day 28). Logistic regression models with generalized estimating equations were used to estimate the relationship between ΔCr, MΔCr, TFXM, BFXM and SAB measurements with rejection. An independence correlation structure was used for MΔCr due to a single measurement per recipient. However, an exchangeable, i.e. equal, correlation structure was used for the ΔCr, TFXM, BFXM and SAB data due to repeated measurements per recipient. Receiver operating characteristic (ROC) curves, along with the area under the curve (AUC), were computed for each predictor (9). A 95% confidence interval for the MΔCr AUC was computed using a normal-theory interval, while the others were computed using a jackknife estimate, which takes into account the repeated observations. Fisher's exact test was used to evaluate a possible association of prior kidney transplantation on the incidence of rejection as well as the severity of rejection. A Pearson correlation coefficient was determined to compare the BFXM channel shifts to the SAB normalized values. Kaplan–Meier survival curves were generated to estimate actuarial graft and patient survival.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

Overall clinical outcomes

Seventy consecutive positive crossmatch recipients were included in the study (Table 1) including 41 High DSA recipients (all recipients of living donor kidneys) and 29 Low DSA recipients (27 living donors and 2 deceased donors). Interestingly, 73% (51/70) of the recipients were female and 47% (33/70) had a prior transplant (range 1–3). Baseline DSA levels as measured by the BFXM ranged from 73 to 565, and 27% (11/41) patients in the High DSA group had a positive T-cell AHG crossmatch at baseline whereas no patients in the Low DSA group had a positive T-cell AHG. All recipients had confirmation of antidonor HLA specificity using SABs.

Table 1.  Demographic data for positive crossmatch kidney recipients included in the present study
 GenderAge at Tx (range)Pre-Tx PP (range)Post-Tx PP (range)Baseline DSA1 (range)Prior renal Tx
MaleFemale
  1. Tx = transplant; PP = plasmaphereses; DSA = donor-specific alloantibody; NA = nonapplicable.

  2. 1Channel shift (B-cell flow crossmatch); 2p < 0.001; 3p = 0.022.

All patients (n = 70)195148 ± 13.3 (15–78)3.8 ± 4.0 (0–15) 9.1 ± 9.2 (0–43)304 ± 107 (73–565)33 
Low level DSA (n = 29) 62353 ± 12.1 (26–78)NA 3.8 ± 6.1 (0–23)203 ± 642 (73–288) 93
High level DSA (n = 41)132844 ± 12.7 (15–65)6.5 ± 3.0 (1–15)12.8 ± 9.3 (0–43) 375 ± 662 (200–565)243

All High DSA patients underwent pretransplant PP (mean number of PP treatments = 6.5 ± 3.0, range 1–15) with the goal of achieving a T and BFXM channel shift <300 (estimated MESF of <19 300) on the day of transplant. Two patients early in the experience did not achieve this endpoint and underwent transplant with BFXM channel shifts of 338 and 328. Neither experienced hyperacute rejection. Another patient was transplanted with a TFXM channel shift of 293 and BFXM of 300 on the day of transplant and developed hyperacute rejection and graft loss (this patient was not included in further analysis for incidence of AHR). The overall incidence of diagnosed AHR was 36% (25/70). AHR was diagnosed in 40% (16/40) of High DSA recipients and in 31% (9/29) of Low DSA recipients. The baseline TFXM did not correlate well with posttransplant AHR (Figure 3A). Baseline BFXM channel shift demonstrated a better correlation with AHR (Figure 3B). AHR rates increased with increasing baseline DSA: 8% (1/12) with BFXM <200; 42% (8/19) with BFXM 200–300; 35% (9/26) with BFXM 300–400 and 54% (7/13) with BFXM >400. All AHR was treated with PP, yet 28% (7/25) developed worsening histologic injury during the treatment course (i.e. C4d deposition alone progressing to rejection or moderate rejection progressing to severe rejection). All AHR rejection episodes were reversible in those patients who regained renal function. However, laparoscopic splenectomy was performed in 10 patients in an attempt to reverse particularly severe AHR (i.e. AHR with rapidly rising DSA, DSA levels resistant to repeated PP or rapidly deteriorating graft function). No patient developed cellular rejection during the first month posttransplant. The actuarial 1 and 3-year graft survival was 91% and 84%, respectively, while the actuarial 1 and 3-year recipient survival was 96% and 94%, respectively. Two recipients died within the first year posttransplant with a functioning allograft.

image

Figure 3. Correlation between the baseline antibody level and the incidence of rejection in the first 28 days posttransplant as measured by T-cell flow crossmatch (A) and B-cell flow crossmatch (B). A channel shift of 300 corresponds to a MESF of approximately 19 300.

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Time course of serum DSA levels

A major goal of this study was to determine the time course of DSA levels after +XM kidney transplant. Thirty-six patients had BFXM data available at baseline, the day of transplant and posttransplant days 4, 10 and 28. These recipients can be stratified into four groups as follows:

  • Low DSA/AHR− Five Low DSA recipients who did not develop AHR had sequential BFXM data (Figure 4A). None of these patients underwent PP and all maintained a BFXM channel shift below 300 throughout the entire 28-day posttransplant period. The BFXM generally was lower than baseline on day 4 and day 28 in Low DSA recipients not developing AHR.

  • Low DSA/AHR+ Seven Low DSA recipients developed AHR (Figure 4B) and had sequential BFXM data. The mean BFXM channel shift in this Low DSA/AHR+ group was 240 ± 30 at baseline, 228 ± 127 on day 4, 486 ± 63 by day 10 and 261 ± 63 on day 28. In 71% of patients (5/7), the BFXM decreased from day 0 to 4, but increased in two patients, one of whom was diagnosed with AHR at this time point. AHR generally was diagnosed by day 10 and was associated with an increased BFXM channel shift (p = 0.008) compared to baseline. PP was performed in all seven patients, but was stopped by day 28. Importantly, the BFXM channel shift was decreased in all seven patients by day 28 suggesting that the increase in DSA may be transient in this group.

  • High DSA/AHR− Twelve High DSA recipients who had sequential data did not develop AHR (Figure 4C). All underwent pretransplant PP, all but one achieved a BFXM channel shift <300 on the day of transplants and all received at least four posttransplant PP treatments. Similar to the Low DSA/AHR-recipients, the BFXM channel shift generally remained low throughout the posttransplant period in this group. By day 4, only one recipient had a channel shift >320. By day 28, 92% of this group (11/12) had BFXM channel shifts <300 and all had BFXM channel shifts lower than baseline. The mean BFXM channel shift in this High DSA/AHR group was 387 ± 63 at baseline, 236 ± 108 on day 4, 211 ± 72 on day 10 and 197 ± 78 on day 28.

  • High DSA/AHR+ Twelve High DSA recipients who had sequential data developed AHR (Figure 4D). All but one of these recipients had a BFXM channel shift <300 on the day of transplant, but five had a BFXM >300 by day 4 despite continued PP and three of these were diagnosed with AHR at this time point. By day 10, 92% (11/12) had BFXM channel shifts >300 and were diagnosed with AHR. Only one patient achieved the maximum DSA level by day 28 and was diagnosed with AHR at this later time point. High DSA/AHR+ recipients tended to have more persistently high DSA with only 3/11 with BFXM <300 by day 28. Five patients in this group underwent splenectomy secondary to a refractory rejection episode (severe histologic injury with high antibody levels) who did not respond to treatment. The mean channel shift for recipients who received a splenectomy at 28 days posttransplant was 353 ± 94.2 compared to a mean channel shift of 301 ± 103.6 for those recipients who responded to apheresis.

image

Figure 4. DSA levels from baseline to 28 days posttransplant for the Low DSA patients without AHR (A) Low DSA patients with resulting AHR (B) High DSA patients without AHR (C) and High DSA patients with resulting AHR (D). Patients who underwent splenectomy are identified by an asterisk. A channel shift of 500 corresponds to a MESF of approximately 131 700.

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When the four groups are considered as one, the mean DSA as determined by the BFXM decreased significantly from baseline to day 4 (p = 0.0002). Decreased DSA levels on day 4 were observed in 71% (12/17) patients who later developed AHR. At day 10, the mean BFXM channel shift was 468 in recipients who developed AHR and 201 in recipients without AHR suggesting that AHR was associated with increased DSA levels posttransplant. By day 28, the BFXM was less than 350 in all patients who did not develop AHR and in 58% of recipients (11/19) who developed AHR.

Correlation between DSA levels and histologic changes

Fifty-three of 70 (76%) recipients underwent at least one biopsy within 28 days of transplantation with serum DSA levels measured within 24 h of the biopsy. One patient who developed AHR did not have serum within this time frame and was not included in this sub analysis. A total of 84 biopsies were included for analysis including 55 protocol biopsies and 29 performed for suspected AHR. Of these, 48 showed no AHR; 6 C4d depositions alone without other histologic injury, 17 moderate AHR and 13 severe AHR. Of the six biopsies demonstrating C4d deposition alone, three progressed to AHR on subsequent biopsies. The mean time from transplant to each histologic diagnosis was C4d alone, 6.7 days ± 3.1 (range 4–12); moderate AHR, 11.3 days ± 5.9 (range 4–24) and severe AHR, 9.7 days ± 5.1 (range 4–21). The overall mean time to AHR was 10.5 days ± 5.5.

There was a strong correlation between histologic injury and DSA level as measured by both BFXM (Figure 5A) and total DSA by SABs (Figure 5B). There did not appear to be a clear association between histologic injury and the TFXM (Figure 5C), with 16% (4/25) of AHR episodes occurring when the TFXM was <200. Similarly, neither ΔCr at the time of biopsy (Figure 5E) nor MΔCr during a rejection episode (Figure 5F) correlated well with histologic injury. The ΔCr was less than 1.0 mg/dL in 70% (21/30) of biopsies with histologic evidence of AHR. Furthermore, during the entire rejection episode, in 48% (12/25) of recipients, the serum creatinine changed less than 1.0 mg/dL and in 20% (5/25) of recipients the MΔCr was ≤0.2 mg/dL. In contrast, no patient with a BFXM channel shift <359 (Table 2A) or an MFI below 6234 (Table 2B) had AHR on biopsy (i.e. the false negative rate was zero below this level). A total MFI of 7819 showed the highest combination of sensitivity (0.96) and specificity (0.87), but in the current study using this DSA level as an absolute cutoff would have missed one episode of AHR and falsely identified three others as positive when AHR was not present on biopsy. While MESF intensity was not obtained at the same time as the BFXM, from our retrospective controls we estimate that a BFXM of 359 roughly corresponds to a MESF in the range of 34 000. Given the variability of all alloantibody assays, we suggest that the actual numbers should be considered to represent good estimates of alloantibody levels rather than exact quantifications.

imageimageimageimageimage

Figure 5. Antibody level at time of biopsy associated with severity of rejection as detected by B-cell flow crossmatch (A), single antigen beads (total antidonor HLA antibody; (B) and T-cell flow crossmatch (C). Correlation between the antibody level measured by single antigen beads versus B-cell flow crossmatch (D). Change in the serum creatinine level stratified by rejection severity at the time of biopsy (E) and during the entire rejection episode (F).

Table 2.  Sensitivity and specificity data for B-cell flow crossmatch (A; channel shifts) and SAB (B; total DSA; MFI)
B-cell FXMTrue negativeFalse negativeFalse positiveTrue positiveSensitivitySpecificity
  1. Highlighted data sets represent equal weight given to both sensitivity and specificity. No patient with a BFXM channel shift less than 359 (MESF of approximately 34 000) developed AHR (A) and only two above this level had evidence of AHR on biopsy. Only one patient with a total DSA MFI value less than 7819 had AHR on biopsy (B) and only three above this level had AHR.

A: Channel shifts
 3023509231.000000.79545
 3063608231.000000.81818
 3083707231.000000.84091
 3183806231.000000.86364
 3274004231.000000.90909
 3354103231.000000.93182
 3594202231.000000.95455
 3664222210.913040.95455
 3894321210.913040.97727
 4004331200.869570.97727
 4074341190.826090.97727
 4124351180.782610.97727
 4464361170.739130.97727
 4484371160.695650.97727
 4514381150.652170.97727
 4544391140.608700.97727
 4734310 1130.565220.97727
 4814311 1120.521740.97727
 4854411 0120.521741.00000
 
Total DSATrue negativeFalse negativeFalse positiveTrue positiveSensitivitySpecificity
 
B: Total DSA
 44641509271.000000.62500
 50921608271.000000.66667
 59601707271.000000.70833
 62341717260.962960.70833
 63951816260.962960.75000
 64921915260.962960.79167
 78092014260.962960.83333
 78192113260.962960.87500
 98922123250.925930.87500
 12 3922133240.888890.87500
 13 2732143230.851850.87500
 13 7482153220.814810.87500
 14 0482163210.777780.87500
 14 3612262210.777780.91667
 14 5052272200.740740.91667
 14 5552282190.703700.91667
 14 9982381190.703700.95833
 15 0752391180.666670.95833
 15 8042310 1170.629630.95833

These results suggest that posttransplant serum DSA levels might be used as monitoring tools for AHR. ROC curves were created (Figure 6) to determine the ability of each posttransplant test (TFXM, BFXM, SABs, ΔCr and MΔCr) to predict AHR. As ranked by AUC, the tests that predicted AHR best were (in descending order) BFXM (p = 0.015, AUC = 0.987, CI 0.96–1.0); highest MFI for Class I DSA level + highest Class II DSA level (p = 0.0003, AUC 0.938, CI 0.853–1.0); total antidonor HLA MFI by SABs (p = 0.0168, AUC 0.935, CI 0.84–1.0) and the highest single MFI for any one donor HLA (p = 0.0001; AUC 0.919 CI 0.827–1.0). Since most rejections involved Class I, the TFXM (p = 0.0005, AUC = 0.831, CI 0.703–0.959) also reached statistical significance, but, as noted above, would have missed 16% of AHR episodes. We favor utilizing the total DSA based on observations that a few patients had relatively equal DSA MFIs that when added together appeared to reach a level that was similar to that causing AHR in patients with only one DSA. Thus, adding each of the SAB MFIs together correlated with histology better than any one DSA alone.

image

Figure 6. ROC curves for predicting antibody-mediated rejection: (A) Comparison of TFXM (p = 0.0005; AUC = 0.831 CI 0.703–0.959) versus BFXM (p = 0.015; AUC = 0.987 CI 0.96–1.0); (B) SAB MFI data comparing total DSA level (p = 0.0168; AUC 0.935 CI 0.84–1.0) versus the highest Class I DSA MFI level + the highest Class II DSA MFI level (p = 0.0003; AUC 0.938 CI 0.853–1.0) versus the highest single MFI level of DSA (p = 0.0001; AUC 0.919 CI 0.827–1.0); (C) Maximum delta creatinine during a rejection episode (p = 0.001; AUC 0.946 CI 0.886–1.0) versus delta creatinine at time of biopsy (p = 0.37; AUC 0.841 CI 0.735–0.946).

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Correlation between AHR and serum creatinine

An important clinical question is whether or not a rise in serum creatinine can be used as the primary method of monitoring for AHR. Both the maximum increase in creatinine from baseline in the entire 28 days (MΔCr) and the change in creatinine at the time of biopsy (ΔCr) was determined in each recipient. AHR recipients had a mean MΔCr of 0.04 mg/dL ± 0.09, C4d+ alone biopsies showed a mean MΔCr of 0.467 mg/dL ± 0.40, recipients with moderate rejection had a mean MΔCr of 0.85 mg/dL ± 0.89 and those with severe histologic injury had a mean MΔCr of 2.48 mg/dL ± 1.69 (Figure 5F). As noted above, ROC curves showed that while MΔCr was associated with AHR, the ΔCr level at the time of biopsy failed to reach statistical significance. Thus, serum creatinine levels are a poor screening tool for AHR. The mean MΔCr also was higher in severe AHR compared to moderate AHR suggesting that while this distinction may not reflect differences in DSA levels, it does result in differences in graft dysfunction.

AHR: Impact of antibody class and method of sensitization

At the time of AHR, 64% (16/25) of recipients had DSA against both Class I and Class II. Five recipients (20%) had only anti-Class I DSA and four (16%) had only anti-Class II DSA.

The method of sensitization did not appear to affect the incidence or severity of AHR. Compared to recipients sensitized by other means, recipients with a prior transplant (n = 33, range = 1–3) had similar rates of AHR (47% vs. 57%, p = 0.28) and similar severity score (p = 0.21).

Correlation between BFXM and SAB data

Finally, the concomitant use of FXM and SAB data in the current study provides an opportunity to assess the agreement between these two modalities in determining DSA levels. It also allows for generalization of our local use of BFXM to the more widely used SAB assay. When all time points were assessed for which both BFXM and SABs were performed, there was a strong correlation between BFXM channel shift and total antidonor MFI by SABs (Figure 5D, Pearson correlation coefficient 0.73, SE 0.048, p = 0.001). There appears to be only one instance in which a relatively high BFXM channel shift (583) was found to have a low total MFI (5960).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

The current study demonstrates several important points regarding AHR in +XM kidney transplant recipients: (1) AHR occurs across a wide spectrum of baseline DSA levels as determined by T and BFXM levels including those associated with a negative T-cell AHG crossmatch; (2) the risk of AHR generally increases with increasing baseline DSA levels, but the occurrence is still unpredictable; (3) prior kidney transplant does not increase the incidence or severity of AHR compared to other methods of sensitization and (4) anti-Class II DSA alone (10) or in combination with anti-Class I plays an important role in AHR and may be the sole cause of AHR in up to 16% of cases.

DSA levels decreased significantly by day 4 in all but a minority of +XM recipients and remained low in patients who did not develop AHR. Since desensitization protocols and conventional immunosuppression have little impact on antibody secreting plasma cells (PCs) (11,12), this decrease in DSA is not likely due to decreased antibody production. Rather, the decrease in DSA levels is more likely due to either absorption of DSA by the allograft (13,14) or the development of blocking (antiidiotypic) antibodies (15).

Posttransplant DSA levels increased in patients with AHR. Mechanistically, this might be explained by increased production of DSA via the generation of a memory B-cell response especially in recipients with low levels of DSA at baseline. Thus, according to this model, AHR might be prevented by therapy aimed at blocking memory or removing memory B cells. The increase in DSA often was transient with even high levels returning to pretransplant levels or lower by day 28. This suggests that AHR response was self-limited and the generation of higher levels of DSA during AHR may not result in persistently high levels. This might be explained by findings from previous studies suggesting that newly generated PCs must compete with existing PCs for survival niches in order to become long-lived PCs and that few newly generated PCs persist (16, 17). Thus, it is important to consider the possibility that increased DSA levels may be transient when assessing the efficacy of therapy for the treatment of AHR including splenectomy (18) or rituximab (19).

The current study also suggests that there is a level of DSA above which AHR reproducibly occurs. While the exact number may vary between laboratories (in our laboratory it is a BFXM >359 or approximately 3.5 times the positive cutoff level and correlates a MESF of approximately 34 000), we contend that posttransplant monitoring of serum DSA levels allows for early intervention to either preemptively treat AHR and/or ameliorate antibody-mediated damage. Conversely, these data suggest that serum creatinine is an insufficient monitoring tool in +XM kidney transplant recipients because AHR frequently precedes any elevation in serum creatinine or may occur subclinically (20). The significance of untreated subclinical AHR, however, remains unclear.

Alternatively, the finding that DSA levels initially decrease before rising during AHR might be explained by alterations in the allografts ability to absorb DSA. Allografts that do not develop AHR might be those that effectively absorb the antibody. AHR might lead to endothelial cell injury and decreased allograft perfusion, which then might manifest as increased serum levels of DSA. Thus, in this model, High DSA levels are not the cause, but rather the result, of AHR. More detailed studies are needed to assess the role of the allograft in the determination of DSA levels posttransplant.

The finding that 29 of 30 (97%) AHR biopsies were C4d+ suggests a role for complement in AHR, yet causative evidence is lacking. We found a small number of biopsies that were C4d+ yet had no other sign of injury and these patients had DSA levels in between those of normal biopsies and AHR. Three of these patients progressed to AHR suggesting that C4d deposition could be an early sign of rejection. However, the fact that three other patients had C4d+ biopsies and never progressed to AHR suggests that there is a level of DSA that triggers complement activation, but is insufficient to cause histologic injury visible by light microscopy.

What is clear from the current study is that serum DSA levels have a direct linear relationship with degree of histologic injury of the allograft. Since AHR can occur when only anti-Class II DSA is present, assays that measure only Class I (e.g. the TFXM) are inadequate monitoring tools. Conversely, the BFXM and the total DSA measured by SABs both appear suitable for monitoring AHR with excellent sensitivity, specificity and ROCs. Only one patient demonstrated severe AHR with strongly positive BFXM and only modest levels of DSA by SABs. The most likely explanations for this situation include antibody against non-HLA antigens, antibody against HLA antigens not present on the single antigen beads or erroneous donor HLA typing. It is also noteworthy that despite the possibility that non-HLA antibodies may be important in sensitized patients, in the current study 96% (24/25) of cases, the antibody associated with AHR was specific for an identifiable donor HLA type.

These data also demonstrate good correlation between the BFXM and the SAB assays over a wide spectrum of DSA levels suggesting that in most cases SABs can be used to predict crossmatch results. Since donor lymphocytes are not consistently available, we suggest that daily monitoring of total DSA using SAB analysis is a useful method to monitor for AHR in +XM kidney transplants.

Gebel et al. have suggested that sensitized renal transplant recipients have a spectrum of risk depending on the level of antibody measured at baseline, but they emphasized that the risk is not clearly defined (21). We suggest that it is also important to define what ‘risk’ is being considered (i.e. hyperacute rejection, AHR or chronic transplant glomerulopathy) and whether or not the risk can be modified by desensitization protocols. The risk of hyperacute rejection in patients not undergoing desensitization likely is directly related to baseline DSA levels. However, the risk of hyperacute rejection is modifiable in that it can be decreased with pretransplant PP making the actual risk of hyperacute rejection most closely related to the level of DSA at the time of transplant. The current study suggests that using our current protocol of selective PP, the risk of AHR is not closely related to the baseline level of DSA, but rather to the level of DSA in the early posttransplant period.

Assessing the efficacy of different approaches to sensitized patients can be difficult. We believe that one of the major difficulties arises when there is insufficient antibody characterization of the study subjects to allow comparisons between different protocols—especially when crossmatch data are reported as merely positive or negative and when there is no verification that the antibody is actually DSA. The current study illustrates that the level of DSA (against both Class I and Class II) is an important determinant of both short- and long-term outcomes. Thus, we suggest that it is critically important that future studies of sensitized patients include comprehensive, quantifiable data regarding the total DSA level as determined by BFXM and the MFIs obtained by solid phase assays. The use of standardization methods such as molecules of equivalent soluble fluorochrome units (MESF), while only estimated retrospectively in this study, might be a useful method of comparing results from different laboratories (6).

In conclusion, the current study suggests that increases in DSA levels posttransplant are associated with AHR, but this increase may be transient. We advocate the use of posttransplant monitoring with BFXM or SABs with early intervention to prevent or ameliorate the impact of AHR. However, important questions remain such as whether or not the allograft significantly absorbs DSA and whether or not changes in DSA production play a role in the pathogenesis of AHR.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References