In a recent study, we compared IgG-MFI, C1q-MFI and dilution studies to measure efficacy of desensitization using Rituximab as induction followed by multiple cycles of plasmapheresis/low-dose intravenous immunoglobulin.15 Antibody levels were measured by the three approaches immediately before antibody removal therapy and immediately at the completion of treatment to determine “delta-reduction” of antibody load. Neither IgG-MFI nor C1q-MFI showed consistent reduction of antibody load, leading to the enigmatic observation that different antibodies within the same serum sample respond differently to treatment. Using dilution studies we demonstrated a very tight and consistent reduction of antibody titer/load in line with the expected response to treatment. Hence we concluded that for treatment purposes, measuring antibody load by titration is more accurate and predicable than using MFI units through either the IgG or C1q assays.
POSTTRANSPLANT DE NOVO DONOR-SPECIFIC ANTIBODIES
De novo donor-specific antibodies (dnDSA) can develop at any point posttransplantation and have been associated with a greater risk of graft dysfunction and loss.16,17 Serial serum monitoring in low-risk renal transplant recipients has documented that 2 of 3 dnDSA can be detected in the serum before the development of clinical dysfunction.16 After dnDSA development heterogeneity exists in the progression to clinical dysfunction and graft loss that is partially, but not entirely captured by clinical and pathologic risk factors available when dnDSA develops.18 Using dnDSA titers or MFI to aid risk assessment is intriguing because it is noninvasive, rapid, and relatively inexpensive. In addition, accurate risk stratification would allow physicians to target patients with high-risk dnDSA using more aggressive immunosuppression while sparing those with low-risk dnDSA the toxicity and cost associated with potent immunosuppression.
As mentioned above, MFI has often been used as a surrogate of antibody strength in studies dealing with HLA antibodies, including dnDSA. Unfortunately, studies correlating MFI, C1q, or C3d status with clinical outcomes are almost universally a mixture of pretransplant DSA and dnDSA, making it difficult to understand the significance of dnDSA MFI at the time of its development.19-21
A recent was the first to titrate dnDSA at the time of first development and correlate dnDSA titer with histology, clinical dysfunction, and graft loss.18 dnDSA C1q status was evaluated and EDTA was used to prevent complement inhibition. Testing serum with or without EDTA revealed that 1.2% of all beads (17.2% of dnDSA beads) were affected by complement inhibition with the median MFI increasing from 3659 (range 7–16 475) to 20 275 (range, 9666-24 463) after EDTA treatment. There was a strong correlation (R2 = 0.8) between the mean MFI of EDTA-treated serum and dnDSA titer after serial dilutions of 1:16 (mean MFI 385 ± 512), 1:64 (mean MFI 2037 ± 2595), and 1:024 (mean MFI 14 762 ± 6101). The correlation between MFI and dnDSA titer was lower (R2 = 0.6) when sera was not pretreated with EDTA. Recipients in this study had a progressively higher risk of graft loss (17%, 29%, and 63%) with increasing dnDSA titer of 1:16, 1:64, 1:1024 respectively (P < 0.01). dnDSA titer also correlated with antibody-mediated rejection and T cell mediated rejection in biopsies done at the time of dnDSA development. However, once the time from dnDSA development to graft loss was considered, a Kapan-Meier graft survival analysis showed no correlation between allograft loss and dnDSA titer (P = 0.14). Furthermore, multivariate Cox models of post-dnDSA survival revealed that the clinical phenotype of the recipient (subclinical vs. clinical at the time of dnDSA development) and their medication adherence were the only independent multivariate predictors of graft loss. After adjusting for clinical phenotype and adherence dnDSA titer (ie, titer ≥1:64, or ≥1:1024) was not correlated with graft loss or estimated glomerular filtration rate (eGFR) decline. Interestingly, there were no patients who had both a subclinical phenotype and dnDSA titer less than 1:64 that progressed to graft loss (8/70 dnDSA recipients). Thus, a combination of low titer and subclinical phenotype may predict low risk for graft loss; however, larger cohorts will be required to evaluate this using multivariate models.
An important observation from the above data is that the IgG MFI correlated better with titer and C1q status in the posttransplant dnDSA study compared with the pretransplant and desensitization studies mentioned above. There are multiple reasons that may explain the discrepancy: (a) the study done on posttransplant dnDSA generally deals with much lower titer antibodies (patients were already immunosuppressed) in MFI ranges where the correlation is generally better; (b) in the posttransplant dnDSA study all sera were treated with EDTA to remove complement inhibition whereas the heat inactivation was used in some of the pretransplant sera; (c) the EDTA concentration used in the posttransplant dnDSA study was significantly greater (25 mM vs. 60 mM) which may have resulted in more complete elimination of the complement inhibition.
Although there are no other studies of dnDSA titer correlated with clinical outcomes, MFI has been correlated with outcome in previous reports. DeVos et al.22 studied a cohort of 48 patients with HLA-DQ dnDSA and found no correlation between antibody-mediated rejection or graft loss when dnDSA were categorized as weak (MFI, 2000-4000); moderate (MFI, 4000-8000); strong (MFI, 8000-15 000); or very strong (MFI ≥ 15 000). Similarly, Dieplinger et al23 studied a small cohort (n = 24) of renal transplant recipients and found no significant association with the median MFI at first detection and 25% or greater eGFR decline. Although peak MFI in the first year after dnDSA detection and delta MFI slope over time correlated with eGFR decline, no attempts were made to adjust for the type or duration of treatment recipients may have received post-dnDSA. A common issue in these and other studies is that complement inhibition was not prevented routinely nor titration used to capture the full picture of dnDSA strength.
Although some have argued that the C1q assay (covered in more detail in other reports in this issue) can uncover recipients whose dnDSA are high risk despite a low MFI, recent data indicates patients with low IgG MFI C1q-positive DSA are in fact the beads affected by complement inhibition. In EDTA-treated serum all C1q-positive beads had an MFI of 10 126 or greater (range, 10 126-24 462) and EDTA MFI predicted C1q status with an area under the curve 0.99.18 If sera were not pretreated with EDTA, then the same C1q-positive beads had an MFI range of 7 to 20 853 (median, 7859). Schaub et al24 also used EDTA-treated sera and found MFI to be an excellent predictor C1q status (area under the curve, 0.98). Because most published reports have not used EDTA, dilution, or other mechanisms to prevent complement inhibition it is not surprising that MFI cutoffs that predict C1q positivity have varied from 6237 to 14 154 with a broad range of sensitivities and specificities.24-27 Thus, it is unclear if any meaningful gain in risk prediction is obtained from the C1q assay beyond knowledge of the MFI in EDTA-treated sera or knowledge of the dnDSA titer.
The data presented here emphasize the inaccuracy of MFI as the sole measure of antibody strength and highlight the utility of dilution studies as the best measure of antibody load. As long as complement inhibition has been reduced or prevented, low-level IgG-MFI values provide a reasonable assessment of antibody strength. In the presence of complement inhibition, epitope spread, or bead saturation, the correlation between antibody strength and MFI is significantly jeopardized making it difficult to interpret studies which have tried to correlate MFI and clinical outcomes without addressing these limitations. This study adds to previous evidence suggesting that much of the C1q binding capabilities are associated with increased antibody load (quantity) rather than an inherent quality of the antibody.11,14,28
In the pretransplant assessment, many patients do not require precise antibody titration because the antibody in question is deemed unacceptable. Alternatively, if desensitization is contemplated (eg, highly sensitized patient with a potential living donor) – dilution studies are the only method to capture the true strength of antibodies, providing guidance for the likelihood of effectively reducing antibodies to acceptable levels.15 Furthermore, dilution studies can monitor the efficacy of desensitization or response to treatment in antibody-mediated rejection.
In the posttransplant assessment, it is important to consider dnDSA strength in the context of clinical phenotype, medication adherence, and allograft histology. Recipients presenting with clinical dysfunction and nonadherence at the time of dnDSA development are known to be high risk of progression to graft loss and neither dnDSA MFI nor titer provide additional risk stratification after adjustment for these risk factors.18 Importantly, for both dnDSA and pretransplant DSA, serial measurements of antibody strength to assess therapeutic success necessitate accurate evaluation after taking into account epitope spread, bead saturation, and complement inhibition.
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