Research ArticleImpact of rituximab on the T-cell flow cytometric crossmatch
Introduction
The flow cytometric crossmatch (FCXM) assay is utilized to detect donor-specific anti-HLA antibodies in both solid organ and stem cell transplant recipients [1]. A positive FCXM in the presence of negative complement-dependent cytotoxicity crossmatch (CDCXM) is associated with higher rates of graft rejection and early graft failure [1,2].
Rituximab (RTX) is a genetically engineered recombinant chimeric mouse-human monoclonal antibody (IgG1-k) against CD20 [3,4]. Apart from its use in the treatment of B-cell malignancies [5,6] and autoimmune disorders [7], it is extensively utilized to control and prevent humoral rejection in solid organ as well as haploidentical haematopoietic stem cell transplant [[8], [9], [10], [11], [12], [13]].
Inclusion of B-cell depleting agents like RTX in immunosuppressive protocols along with plasmapheresis/immunoadsorption has allowed renal transplantation across the ABO barrier and expanded the donor pool [[14], [15], [16]]. The long term graft outcome in ABO-incompatible renal transplantation (ABOi-RT) reported by most centres are comparable with ABO-compatible (ABOc-RT) [[16], [17], [18]].
However, RTX when present in the sera of transplant recipients causes false-positive CDCXM (by exerting a cytolytic effect on B-cells) as well as B-cell FCXM (B-FCXM; secondary to recognition by anti-human antibody) [[19], [20], [21], [22]]. Its impact on T-cell FCXM (T-FCXM) has not been reported to date.
Pronase treatment of donor lymphocytes cleaves Fc receptors and thus CD20 from B-cell surface, thereby removing the RTX effect and facilitating the correct interpretation of the B-FCXM [22,23]. The Luminex based tests are not affected by RTX, and to rule out the presence of donor-specific antibodies (DSA) more definitively, a virtual crossmatch using the single antigen bead (SAB) assay is done alongside. Testing of historic serum collected before RTX therapy is another feasible option to rule out DSA [19].
The aim of this study was to evaluate the effect of RTX on T- and B-cell FCXM using Pronase (P-) and Non-pronase (NP-) treated donor lymphocytes and compare it to the SAB assay.
Section snippets
Patients
This is a retrospective analysis of 26 ABOi-RT and 2 haploidentical stem cell transplant recipients on RTX therapy whose samples were received for pre-transplant workup in the HLA and Immunogenetics laboratory in the Department of Transfusion medicine & Immunohaematology between December 2015 to February 2020.
As part of Pre-transplant workup, FCXM using P- and NP- treated donor lymphocytes and SAB assay was performed for all recipients treated with RTX.
Cell isolation
Donor lymphocyte isolation was done by
Patients
A total of 28 patients (26 renal and 2 stem cell transplant recipients) on RTX therapy were analyzed during the study period. The mean age of recipients was 33.5 ± 10.5 years with a male to female ratio (M: F) of 3:1. The pre-transplant evaluation was done against 30 donors (2 donors each for 2 stem-cell transplant recipients). The mean time of testing post-RTX therapy was 21 days. Further demographics are given in Table 1.
For each recipient, B- and T-FCXM using NP- and P- treated donor
Discussion
RTX has replaced splenectomy in most of the immunosuppression protocols for ABOi-RT due to its B-cell depleting action [15,16,24,25]. It is also indicated in induction chemotherapy of highly sensitized transplant recipients to reduce panel reactive antibody (PRA) [[26], [27], [28]] as well as to treat humoral allograft rejection [10,[28], [29], [30]]. Its half-life is between 1.6 and 20 days but is relative to dose and frequency of administration. It can be detected in the serum of transplant
Conclusion
Though traditionally RTX is described to impact B-FCXM only, we describe a consistent finding of ‘T-cell tailing’ phenomenon in 57% of total FCXMs, which clears with pronase treatment. The absence of DSA in all these recipients reinforces the fact that this is a false positive finding and should not be misconstrued as a possible class I donor-specific antibody. To our knowledge, this is the first study to report the effect of RTX on T-FCXM.
In the light of the growing use of RTX in various
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Declaration of Competing Interest
All authors declare that they do not have any potential conflict of interest that could inappropriately influence the present study.
Acknowledgements
None.
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2021, Transplant ImmunologyCitation Excerpt :Complement-dependent cytotoxicity and FCXM for such patients could be considerably influenced. These methods are difficult because of nonspecific antibody binding to Fc receptors on B cells, which is associated with false-positive results. [19] After transplantation, FCXM has not been used for annual monitoring due to a problem with availability of intact donor PBMC.
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First co-authors; Contributed equally.