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Keywords:

  • HLA-specific antibody;
  • kidney transplant;
  • Luminex;
  • molecules of equivalent soluble fluorochrome (MESF)

Abstract

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

Approximately 25% of patients who undergo kidney transplantation develop HLA-specific antibodies, the strength of which has not been previously correlated with graft failure. The strength of these de novo antibodies is investigated in this study. Serial dilution of strong HLA-specific allo-antibodies (up to 1:25,600) and testing with HLA-antigen-coated beads showed that the titer of the reaction to different HLA antigens is directly correlated to maximum fluorescence values and the molecules of equivalent soluble fluorochrome (MESF) values obtained by Luminex machines. Thus, the strength of antibodies can be measured utilizing maximum fluorescence and MESF. The strength of antibodies in the sera from 39 patients who subsequently had graft failure were markedly higher than those in the sera of 26 patients who continued to have good graft function (p = 0.0084). A clear increase in the strength of antibodies was identified in nine patients with a subsequent increase in serum creatinine levels. If analyzed for donor specificity, a strong association was noted for donor-specific MESF and failure (p = 0.00000027). Our results suggest that it is important to monitor the strength of antibodies when evaluating patient sera posttransplant.


Introduction

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

HLA-specific antibodies found in posttransplantation patients have been shown to be strongly associated with graft failure (1). However, using the newer quantitative methods now available, no study has been done showing that increasing strength (titer) of specific antibodies that develop de novo after transplantation leads to the final development of graft failure. We document here the finding that when HLA-specific antibody appears in the peripheral circulation after transplantation, it often increases in strength with time. This finding is possibly of importance in gauging at what point the patient is in the humoral response continuum and how imminent the time of failure may be.

Luminex single antigen bead reactions were first normalized for the quantity of antigens per bead by reaction with the monoclonal W6/32 antibody. The reactions were then compared with standard beads to derive the molecules of equivalent soluble fluorochrome (MESF) values (2).

Patients and Methods

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

Patients

We examined the sera of 65 patients who had functioning kidney grafts that were performed and followed at the University of Miami/Jackson Memorial Medical Center (JMMC). All patients received first kidney transplants from either living related or deceased donors between 1991 and 1999. Of the 65 patients, 39 patients had grafts that functioned for over 1000 days, but were subsequently rejected (failure group). The sera from the 39 patients whose grafts failed were drawn before graft failure. Sera were also drawn from a matched set of another 26 patients who received transplants within the same time frame and retained functioning grafts up to 2003. The average observation period was 2122 ± 710 days in patients who rejected their grafts and 2626 ± 590 days in patients with functioning grafts. There were no statistically significant differences among groups in terms of deceased/living related donor, recipient age, recipient sex, HLA mismatch, immunosuppression regimens and serum creatinine levels at discharge and 6 months after transplantation. Patients were excluded from the study if they died with a functioning graft, had recurrent disease, were known to be non-compliant or had pre-transplant HLA-specific antibodies. Details of patient characteristics were described earlier (3,4). All rejections were subsequently confirmed by biopsy. Unfortunately, C4d staining was not used for biopsies during the major part of the study period, and a retrospective analysis was not possible.

A total of 679 samples (405 samples from the 39 patients in the failure group and 274 samples from the 26 patients in the functioning group) were tested retrospectively. For practical reasons, in this study, we focused on the last-drawn serum sample of each patient (39 samples in the failure group and 26 samples in the functioning group).

Anti-HLA-specific IgG antibodies

Screening tests for anti-HLA-specific IgG antibodies were performed using LABScreen® assays, based upon the manufacturer's instructions (One Lambda Inc., Canoga Park, CA). Briefly, the beads of LABScreen were coated with HLA antigens. In LABScreen assays, antibodies are detected by the fluorescence intensity (FI) of each HLA antigen coated bead. The signals were then normalized using the following procedure. Raw FI data were adjusted for background signal using the formula: [(Sample #N beads – Sample negative control beads) – (Negative control #N beads – Negative control beads)]. The adjusted reaction values for HLA class I specific antibody were additionally normalized using the FI values of the W6/32 monoclonal antibodies tested with the same beads. Details were described in the report of El-Awar et al. (5).

We focused on FI in this study and used the final FI of each bead, which was normalized.

Data acquisition and conversion to MESF values

To convert FI values into MESF values, we used standard beads labeled with fluorescence having different linker lengths, obtained from Bangs Laboratories, Inc. (Fisher, IN). The Quantum™ PE MESF kit (827A) is composed of a set of five populations of calibrated fluorescent standards (500–50000 MESF range): four populations of microbeads having different levels of phycoerythrin (PE) fluorescence intensity and one Certified Blank™ population. The kits were used for quantization of the FI in terms of the number of MESF.

MESF standard beads of the MESF kit (827A) were tested using a Luminex machine. Reaction strength data were obtained as FI values. Each test was done in triplicate. The data was then converted to MESF values by extrapolating from a standard curve. To generate the standard curve, the results of five MESF standard beads with known PE fluorescence values (ranging from 0 to 50,000) were plotted (FI vs. MESF) using the linear regression analysis software Statview (SAS institute, Cary, NC).

Statistical analysis

Significance between frequencies was determined by chi-square analysis or Fisher's exact test. Averages were compared using the unpaired Student's t-test and regression analysis to determine the relationship between the variables. P values <0.05 are reported as statistically significant.

Linear regression analysis was calculated using Statview software (version 5.0.1).

Results

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

Standardization of Luminex fluorescence intensity values

The standard calibration curve of MESF values against FI values was obtained by the linear fitting of the mean values for the four microbeads populations as seen in Figure 1. The following formula derived from the figure was used to convert the FI values to MESF values:

  • image

where Y = MESF, X = FI.

image

Figure 1. Maximum FI of anti-HLA-specific antibodies and MESF. The relationship between FI and MESF is established with the following formula: inline image, where Y = MESF, X = FI.

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Peak MESF and dilutions

Figure 2 provides examples of the MESF values for LABScreen PRA beads with serial dilutions of two strong antibodies. The beads were analyzed according to the strength of MESF. The reaction of every tenth bead is shown in the dilution plots. As can easily be seen, beads with a high titer tend to have a high MESF value in both of these examples, despite a difference in the dilution characteristics of the two sera. This relationship was positive for all of the beads tested. Given these results, we propose that the MESF values derived from the FI be used in the first dilution of an antiserum as a measure of its strength, rather than the titer.

image

Figure 2. Examples of MESF of anti-HLA-specific antibody with dilutions. Two sera with high titers of HLA-specific antibodies are tested in dilutions with screening beads at each dilution. The reactions of every tenth bead are shown here. Note that the bead with the highest titer tended to have the highest MESF value. (a) An example of serum reacted to a dilution of 1:25600. (b) A different serum with slightly different dilution characteristics, but note the same relationship between high MESF and titer.

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Anti-HLA-specific IgG antibodies in sera of patients who subsequently failed compared with sera of patients with continuing graft function

Figure 3 shows the highest MESF values of LABScreen PRA beads for each patient, sorted according to MESF values. The average MESF value was 241,473 for the 39 patients in the failure group and 31,174 for the 26 patients in the functioning group.

image

Figure 3. Maximum MESF of anti-HLA-specific antibodies for the failure and functioning groups. The maximum MESF value found for each patient is plotted as a vertical bar. Note that the patients in the failure group who subsequently had rejected transplants had antibodies of higher activity than the patients with transplants with continued functioning.

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The sera of 10 normal healthy males were analyzed as a control. The sera of this control group have FI values of 0 in all beads after normalization.

Using a cut-off line (the average + SD of the functioning group), we divided each group into high MESF (15 in the failure group and 2 in the functioning group) and low MESF groups (24 in the failure group and 24 in the functioning group). The frequency between the failure and functioning groups was statistically significant (p = 0.0084). The frequency of high MESF yields the conclusion that high MESF is statistically correlated with poor graft function.

Correlation of peak MESF and serum creatinine

Figure 4 shows the highest MESF values of the highest reacting Single Antigen Bead (One Lambda Inc.) tested with sera from patients who rejected their grafts along with their corresponding serum creatinine levels (mg/dL). Nine patients in the failure group had HLA-specific antibodies with increasing MESF values posttransplantation. Representative plots for four of these patients are shown in Figure 4. As can be seen, there is a strong relationship between the high levels of anti-HLA-specific antibody and increasing serum creatinine. The nine patients in the failure group had positive reactions to donor-specific antigen coated beads or epitope-related donor-specific antigen coated beads. In contrast, only one patient in the functioning groups had donor-specific antibody. In both groups, patients with donor-specific antibodies had increasing MESF levels during the transplant period.

image

Figure 4. Example of MESF of the anti-HLA-specific antibody and serum creatinine in the failure group. The MESF for the anti-HLA-specific antibody of each patient and annual serum creatinine was plotted after transplantation. MESF and serum creatinine of each patient increased up until graft failure. (a) This patient had increasing serum creatinine and MESF of donor-specific antibodies before failure and lost the graft at 5.3 year after transplant. Single Antigen Bead testing showed donor-specific antibodies (A31 and DR7). (⋄–⋄): MESF value of anti-DR7-specific antibody, (□–□): MESF value of anti-A31-specific antibody, (▴–▴): serum creatinine value (mg/dL). (b) This patient had increasing serum creatinine and MESF of donor-specific antibodies before failure and lost the graft at 7.7 year after transplant. Single Antigen Bead testing showed donor-specific antibody (A24 and DQ9). (⋄–⋄): MESF value of anti-DQ9-specific antibody, (□–□): MESF value of anti-A24-specific antibody, (▴–▴): serum creatinine value (mg/dL). (c) This patient had increasing serum creatinine and MESF of donor-specific antibodies before failure and lost the graft at 6.2 year after transplant. Single Antigen Bead testing showed donor-specific antibodies (DQ9 and DR4). (⋄–⋄): MESF value of anti-DQ9-specific antibody, (□–□): MESF value of anti-DR4-specific antibody, (▴–▴): serum creatinine value (mg/dL). (d) This patient had increasing serum creatinine and MESF of donor-specific antibodies before failure and lost the graft at 7.5 year after transplant. Single Antigen Bead testing showed donor-specific antibodies (A3 and DQ7). (⋄–⋄): MESF value of anti-DQ7-specific antibody, (□–□): MESF value of anti-A3-specific antibody, (▴–▴): serum creatinine value (mg/dL).

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Donor-specific antibody and MESF values

In the failure group the average MESF value was 606,766 for the 9 patients with donor-specific antibodies. The highest donor-specific antigen was used in these calculations. Using the cut-off line (the average + SD of the functioning group), we divided the 9 antibody-increasing patients of the failure group into either the high MESF group (n = 9) and the low MESF groups (n = 0), and also divided the 26 patients of the functioning group with using the same cut-off line (the high MESF group: n = 2, the low MESF group: n = 24). The frequency of the higher/lower MESF between functioning group and 9 antibody-increasing patients of the failure group was statistically significant (p = 0.00000027).

Discussion

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

The use of beads with standard fluorescence was introduced by Schwarz et al. as a tool to compare quantitative FI measurement over time and across platforms (2). We describe here the use of the MESF procedure on Luminex beads, which should allow comparison of maximum fluorescence values detected in various other laboratories. The importance of measuring the strength of antibodies became apparent when we restudied the sera of patients previously reported (3).

In our previous report, we demonstrated that fewer patients with functioning grafts had antibodies compared with those who subsequently had failed grafts (3). We now report that there was a marked difference in strength of antibodies found in those with functioning grafts compared with those whose grafts failed (Figure 3). The significance level was high if we computed the percentage of patients with high MESF in the failure versus functioning group (p = 0.0084). In our previous study, one patient was shown to have increasing antibody (3). We now see that this may have been a common occurrence among most of the patients who had rejected their grafts.

The increase in antibody is also apparent, as seen in Figure 4, which shows that antibodies increase in strength (titer) with time until the point of graft failure. There were also other five cases in which antibody increase was correlated with increased creatinine levels.

When assessing the strength of a serum, questions have arisen as to which bead should be considered. For practical purposes, the answer to this question may be the strongest bead, since there appears to be a continuum. Donor-specific antigen beads could also be considered to be the most important since 9 patients in the graft failure group had donor-specific antibodies. However, specificities other than donor specific also often appear because of reactions due to sharing of HLA epitopes, as recently noted (6).

The strength of antibodies may also be important in the sera of patients before transplantation. First, strong antibodies may be very difficult to remove if antibody reduction by plasmapheresis or rituximab treatment is initiated, as previously noted (7–9). It is likely that the strength of antibodies is also important if a decision is made to cross a positive cross-match detected by flow cytometry. Weak antibodies may be dealt with by strong immunosuppression, whereas high titer antibodies may be very difficult to overcome.

Because of the expense, all antibodies to be studied could not be titrated. Fortunately, as seen here, the titer is correlated to the maximum fluorescence value obtained from a single test (Figure 2).

Once standardized with MESF values, different laboratories can compare results with other laboratories.

In conclusion, the strength (titer) of antibody present in the sera of transplant patients is a valuable parameter to be considered when studying antibody response post-transplantation.

Acknowledgments

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

The work for supported by grants from Terasaki Foundation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • 1
    Terasaki PI, Ozawa M. Predicting kidney graft failure by HLA antibodies: A prospective trial. Am J Transplant 2004; 4: 438.
  • 2
    Schwartz A, Gaigalas AK, Wang L, Marti GE, Vogt RF, Fernandez-Repollet E. Formalization of the MESF unit of fluorescence intensity. Cytometry B Clin Cytometry 2004; 57: 16.
  • 3
    Mizutani K, Terasaki P, Rosen A et al. Serial ten-year follow-up of HLA and MICA antibody production prior to kidney graft failure. Am J Transplant 2005; 5: 22652272.
  • 4
    Ciancio G, Contreras N, Esquenazi V et al. Kidney transplantation at the University of Miami. In: CeckaJM, TerasakiPI (eds). Clinical Transplants. Los Angeles , CA : UCLA Immunogenetics Center, 1999: 159172.
  • 5
    El-Awar N, Lee JH, Tarsitani C, Terasaki PI. HLA class I epitopes: recognition of binding sites by mAbs or eluted alloantibody confirmed with single recombinant antigens. Hum Immunol (in press).
  • 6
    Cai J, Terasaki PI, Mao Q et al. Development of nondonor-specific HLA-DR antibodies in allograft recipients is associated with shared epitopes with mismatched donor DR antigens. Am J Transplant 2006; 6: 29472954.
  • 7
    Zachary AA, Montgomery RA, Ratner LE et al. Specific and durable elimination of antibody to donor HLA antigens in renal-transplant patients. Transplantation 2003; 76: 15191525.
  • 8
    Vieira CA, Agarwal A, Book BK et al. Rituximab for reduction of anti-HLA antibodies in patients awaiting renal transplantation: 1. Safety, pharmacodynamics, and pharmacokinetics. Transplantation 2004; 77: 542548.
  • 9
    Stegall MD, Gloor J, Winters JL, Moore SB, Degoey S. A comparison of plasmapheresis versus high-dose IVIG desensitization in renal allograft recipients with high levels of donor specific alloantibody. Am J Transplant 2006; 6: 346351.