Abstract
Postoperative complications place a significant burden on patients, with approximately 10-15% of patients experiencing an adverse event. Surgery induces an immune response that, if not properly controlled, can contribute to these adverse events. Monocytes and their surface receptors undergo perioperative-specific phenotypic changes linked to postoperative complications, making them important targets for immune monitoring. Current methods for immune monitoring during the perioperative course utilise spectral flow cytometry using fresh blood for real-time (RT) phenotyping. However, there are challenges, as fresh samples limit recruitment and scalability due to the time taken to process them. Spectral flow cytometry of whole blood stabilisation (WBS) is an effective method for immune monitoring during the perioperative course due to its time-efficient protocol. Enabling cell fixation and marker preservation, WBS allows for retrospective phenotyping across multiple donors. However, optimisation is needed to ensure that frozen, stabilised samples achieve adequate marker resolution, allowing for accurate identification of monocytes and the examination of their function. This project aimed to optimise a 19-colour monocyte panel in spectral flow cytometry WBS for comparable marker resolution to RT-phenotyping with fresh blood samples, to enable reliable immunophenotyping of monocytes during the perioperative course.
Two commercial whole blood stabilisers (Cytodelics, Cytodelics AB, Sweden and PROT-1, Smart Tube, USA) were tested initially for their use in monocyte immunophenotyping. Isolation of leukocytes from the favourable stabiliser was further optimised by reducing RBC aggregation to leukocytes. The flow cytometry panel was then optimised by adding markers to refine the gating strategy, titrating and re-titrating markers, and testing alternative fluorophores to enhance marker resolution. The final protocol was applied to a surgery donor sample and compared with matched RT-phenotyping to evaluate concordance across these two sample types.
PROT-1 was identified as the optimal stabiliser for preserving monocyte marker expression, which was comparable to RT-phenotyping. Optimising the RBC lysis protocol reduced RBC aggregates from 32% to 4%, resulting in a cleaner leukocyte population for downstream gating. Refinement of the panel included the addition of the neutrophil marker CD15, which enabled cleaner discrimination of TLR2+ monocytes and TLR2(dim) neutrophils, and therefore more accurate monocyte gating.
Re-titration of TLR2 and testing alternative fluorophores on CD16 further improved the discrimination of monocytes and their subsets. Using the optimised protocol, stabilised samples successfully reproduced perioperative immune trends seen with RT-phenotyping, including reduced HLA-DR and increased CD163 expression on post-operative day 1 (POD1) compared to the pre-surgery timepoint.
The reliance on fresh blood samples constrains longitudinal immunophenotyping. Overall, we optimised a WBS protocol to enable monocyte phenotyping comparable to RT-data. We demonstrated that optimised protocols could capture clinically relevant immune dynamics, enabling feasible, high-quality longitudinal immunophenotyping in surgical patients.