Preformed antibodies directed against donor human leukocyte antigen (HLA) antigens represent a major obstacle in kidney transplantation, limiting both access to transplantation and kidney allograft survival (1, 2). It is widely accepted that kidney transplantation across donor-specific antibodies (DSA) identified either by solid-phase assays or flow cytometry crossmatch (FCXM) is associated with a higher risk of antibody-mediated rejection (ABMR) and inferior allograft outcomes, even in absence of positive complement-dependent cytotoxicity crossmatch (complement-dependent cytotoxicity crossmatch [CDC XM]) (3–7). Several transplant programs have implemented peritransplant desensitization regimens using T- and B-cell depleting antibody induction, peritransplant apheresis, and high-dose intravenous immunoglobulin (IVIg) to counteract the deleterious effects of preformed DSA (8). Despite intense strategies of desensitization, there is still an increased rejection risk, which may critically depend on the strength of the preformed DSA. Previously, the Viennese group used anti-thymocyte globulin (ATG) induction and peritransplant immunoadsorption (IA) as desensitization regimens in DSA-positive deceased donor kidney transplantation. The only predictor of antibody-mediated rejection found by this study was donor-specific antibody mean fluorescence intensity (DSA MFI) (9). FCXM may have several advantages over DSA MFI in terms of better predictive power to select grafts at risk of ABMR (10, 11). Moreover, kidney transplantation with a low level of DSA with or without a low positive B-cell FCXM was found to be associated with satisfactory outcomes in highly sensitized mostly living donor kidney transplant recipients who received depleting antibody induction and frequently also desensitization (12). In HLA incompatible deceased donor kidney transplantation with peritransplant desensitization, outcome predictors are poorly understood. Therefore, in this retrospective single-center observational cohort study, we assessed several variables to predict antibody-mediated rejection in those DSA positive deceased donor kidney transplant recipients who had undergone peritransplant desensitization.

Kidney transplantation across the HLA barrier is associated with an inferior allograft outcome. It is widely acknowledged that the presence of preformed DSA before transplantation increases the probability of ABMR occurrence (12–15). Strategies, namely, peritransplant desensitization, were implemented in many centers to prevent such an adverse outcome (17–19).

In this retrospective single-center analysis, we evaluated the potential of several clinical and immunological risk factors to predict ABMR in patients who had received deceased donor kidney allograft and in whom the peritransplant desensitization was applied due to the current presence of donor-specific antibodies. We found that pretransplant FXCM and the number of DSAs, but not MFI, are the most reliable tools for ABMR prediction.

It has been widely accepted that the prognostic value of DSA is limited (20, 21). First, Luminex-based DSA determination shows HLA antibody specificity to antigen but no information can be provided in regardsto epitope specificity (22, 23). Second, the Luminex method is unsuitable for exact value measurement because its MFI values are semiquantitative, not presenting the exact titer of antibodies; furthermore, the prozone effect must be taken into account, non-anti-HLA antibodies may interfere with the beads, and the previously mentioned epitopes may be shared between the beads (24, 25).

In many centers, the FCXM assessment is not implemented as a 24/7 service, and therefore, there is a lack of information on whether FCXM outperforms DSA-based risk stratification. Thus, studies on FCXM are not consistent in respect of graft outcomes (10, 26, 27). Our data are similar to Couzi et al. (10), where higher rejection occurrence in patients with both DSA and FCXM positivity prior to transplantation was observed.

Our data from protocol biopsies suggest future long-term outcomes to be inferior as patients with positive pretransplant FCXM exhibit frequent subclinical rejections in 3-month protocol biopsies. However, it is well known that sensitized patients who have undergone desensitization and HLA incompatible living donor kidney transplantation have substantial survival benefits compared to those patients who did not undergo transplantation and those who waited for transplants from deceased donors (28). However, the overall incidence of ABMR in those patients was high when 24 out of 267 patients developed severe oliguric early ABMR treated with eculizumab or splenectomy and transplant glomerulopathy occurred later (29).

Data on HLA incompatible deceased donor kidney transplantation are scarce. In their pioneer work, the Vienna group evaluated the outcomes of 101 HLA incompatible kidney transplantation (9) who had undergone IA-based desensitization prior and after transplantation along with rabbit anti-thymocyte globulin. Most DSA+ patients had a negative CDC XM already before IA. Three-year death-censored graft survival in DSA+ patients was 79 and 33% of patients experienced ABMR. The authors described only a trend toward higher ABMR rates in positive baseline CDCXM while those patients with DSA MFI >15,000 experienced ABMR in 71%. Similarly to our study, Amrouche et al. (30) described in 95 patients, who received similar posttransplant desensitization due to DSA MFI levels >3,000 while negative complement-dependent cytotoxicity-negative crossmatch, satisfactory long-term outcomes: the 3-year death-censored allograft survival rates were 91%, and recipient survival rates were 93%, respectively. Those data are similar to our FCXM-negative cohort. Of note, the incidence of ABMR in the Amrouche study remained high and was detected in 32% of recipients which is similar to our FCXM negative cohort. Contrary to expectations, in our study, the FCXM positive cohort presented with far poorer outcomes in terms of ABMR incidence and 3-year graft outcomes. Therefore, it is likely that our HLA incompatible cohort was at a far higher risk in comparison with others. This fact also points out the necessity of more advanced risk stratification ahead of transplantation which may allow successful kidney transplantation even in patients with DSA MFI over 5,000. Our study shows the association of pretransplant FCXM with ABMR also in patients with MFI <5,000 or with class I DSA. Therefore, it is likely that pretransplant FCXM positivity outperforms any DSA level from historical sera.

Based on the presented data, we have already modified a pre-transplant risk assessment in our center. In all kidney transplant recipients with present preformed DSA, the positive FCXM prior to transplant represents a veto for transplantation. Unpublished data suggest on far lower incidence of acute ABMR when this approach was implemented. It is, however, also likely that those patients with repeated positivity of FCXM prior to transplant are being trapped on the waiting list for a significant period of time, although the general waiting time to transplant is much shorter in comparison to other countries (31). Of note, described the poor outcome of HLA incompatible transplantation with pretransplant FCXM positivity justifies longer waiting time for more compatible donors as there is a lack of available organs and better allocation would finally increase the patient-years with functioning grafts. Some of those sensitized patients may also find their compatible donor in the case of kidney pair donation when a living donor is available (32).

The limitation of this study is the lack of DSA DP and DQ assessment, as donor DP and DQ typing were not available in the whole cohort.

In conclusion, our data suggest that pretransplant FCXM and DSA number, but not DSA MFI, are independent predictors of ABMR in patients who received peritransplant desensitization. We, therefore, suggest the implementation of FCXM assessment in a daily routine ahead of DSA positive deceased donor kidney transplantation when peritransplant desensitization is planned.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by the Ethics Committee of the Institute for Clinical and Experimental Medicine and Thomayer Hospital (listed as No. A-19-24). The patients/participants provided their written informed consent to participate in this study.

OV and GB designed the research. KO, GB, and OV wrote the manuscript. KO and PH collected the data. KO, OV, JS, AS, JM, and TM performed the research. KK provided the FCXM sera analysis. KO, PH, and JK participated in the data analysis and OV supervised the research. All authors contributed to the article and approved the submitted version.

This study was supported by the Ministry of Health of the Czech Republic MZO 00023001 and by the Ministry of Health of the Czech Republic under grants NV19-06-00031 and NU21-06-00021. The authors wish to thank the staff of Immunogenetics Laboratory and Transplant Laboratory for their valuable help with FCXMs evaluation. They are also grateful to Michael Fitzgerald for the English correction.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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