STANDARDIZED IMMUNOPHENOTYPING IN THE CANADIAN NATIONAL TRANSPLANT RESEARCH PROGRAM: PILOT TRIAL OF POST HSCT SAMPLES AND DEVELOPMENT OF AUTOMATED GATING PIPELINES

Sabine Ivison ^0,0; Mehrnoush Malek ⁰; Rosa Garcia ^0,0; Raewyn Broady ^0,0; Anne Halpin ⁰; Manon Richaud ⁰; Rollin Brant ⁰; Jean-Sebastien Delisle ⁰; Lori West ⁰; RyanR. Brinkman ⁰; Megan Levings ^0,0; Salima Janmohamed

⁰Division of Surgery, Department of Medicine, University of British Columbia, Vancouver, BC, Canada; ¹Beckman Coulter Life Sciences, Division of Beckman Coulter, Inc., Miami, , ; ²BC Cancer Research Institute, University of British Columbia, Vancouver, BC, Canada; ³Alberta Transplant Institute, University of Alberta, Edmonton, BC, Canada; ⁴Hopital Maisonneuve-Rosemont, University of Montreal, Montreal, BC, Canada; ⁵BC Children's Hospital Research Institute, University of British Columbia, Vancouver, BC, Canada

Background: Immune monitoring of post-transplant blood by flow cytometry (FCM) can identify features of immune tolerance and may help predict rejection. Discovery and validation of clinically useful biomarkers requires the analysis of large numbers of samples. Incorporating standardized FCM panels into clinical research enables the merging of data across time, site and study. We previously established standardized immunophenotyping at several sites across Canada using blood from healthy donors, and have now applied these protocols to the analysis of post hematopoietic stem cell transplant (HSCT) samples and added automated data analysis pipelines.

Methods: Peripheral blood from 11 HSCT recipients 100d post transplant was analysed within 4h of collection using six DuraClone IM dry format panels originally developed by The ONE Study1. Blood was aged for 24 h prior to isolation and freezing of PBMCs, and distributed to three sites for comparative analysis. Relative biological variation was expressed as intraclass correlation coefficients (ICCs). Results from central manual analysis were compared to those obtained by supervised, flowDensity-based automated gating pipelines.

Results: FCM of identical, 24 h-aged and thawed PBMCs from post HSCT patients at 3 sites yielded very similar data (ICC>0.75 for most values); ICC values were not significantly lower than those obtained with freshly frozen PBMCs from healthy donors, with the exception of some monocyte and B cell subtypes. Automated gating pipelines were created to extract data from healthy and post HSCT samples that fell within the range defined by three independent manual analyses. Finally, merging of phase 1 (healthy donors) and phase 2 (100 d post HSCT patients) studies revealed that monocytes were increased and lymphocytes (especially B cells) were decreased in the latter; B cells were shifted in favour of immature/transitional populations, while no significant differences were seen in the T cell populations.

Conclusions: Standardized immunophenotyping can be successfully applied to notoriously difficult (aged & thawed post-transplant) samples. Whole blood-derived data obtained in separate, multi-site studies can be merged and automated gating pipelines can provide accurate and robust analysis of blood from very different donor types. Thus, this technology is suited to large-scale studies for the discovery and validation of clinically relevant biomarkers.