Purpose: Donor-specific antibody (DSA) and antibody (Ab)-mediated rejection are challenges in long-term outcomes and limit re-transplantation. Sensitizing events such as homografts in surgical procedures, assist device implantation and transfusions may lead to sensitization, transient immune activation or passive transmission of HLA-Ab. Unique to children, thymectomy is routinely performed during cardiac transplantation or prior surgery. Our overarching goal is to study the impact of thymectomy on development of de-novo DSA. Here, as a first step we evalulated mixed lymphocyte reaction (MLR) alloreactive T cell proliferation as combined with standardized flow cytometry phenotyping (SFC; Duraclone IM, Beckman Coulter), used in the Canadian National Transplant Research Program and the ONE Study to standardize data across sites.


Methods: MLR and flow phenotyping were performed using control adult (n=9) and pediatric (n=3) peripheral blood mononuclear cells and irradiated pooled HLA mismatched 3rd-party splenocytes. Cell proliferation was detected using CellTrace Violet dye combined with the SFC panel or BrdU incorporation ELISA (n=12). Pre- and post-transplant DSA data from our clinical lab were collected (n= 117 patients).

Results: Proliferation in response to mismatched HLA was seen in the BrdU assay, but this lacked the ability to identify which cells proliferated. Proliferation dye was readily detected in the SFC panel. Unstimulated cells did not proliferate whereas mitogen stimulation yielded strong proliferation. Phenotypic changes comparing pre- to post-MLR analysis included decreased %T cells (although CD4 and CD8 remained consistent), increased %B cells, increased %NK cells, decreased %NKT cells and disappearance of monocytes. Samples of 0.2[times]10<sup style="font-family: Verdana;">6 cells were sufficient for phenotyping. 
Evaluation of de-novo DSA frequency is ongoing.

Conclusion: Our preliminary results show the SFC panel in combination with proliferation dye is a novel way to refine the detection of cell proliferation in MLR assays. Standardized phenotyping can allow the detection of alloimmune responses in individual patients in a reproducible manner between patients and centres, and provide an opportunity to standardize clinical investigation of immune responses.

CITATION INFORMATION: Halpin A, Sosniuk M, Urschel S, Campbell P, Larsen I, West L. Refinement of an Old Faithful: A Standardized Flow Cytometry Approach to Measure Donor-Recipient Alloreactivity (a Pilot Study). Am J Transplant. 2017;17 (suppl 3).

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