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ThesisCC BY-NC V4.0, Open Access
Thesis
Evaluating the monocyte monolayer assay: assay conditions, inflammatory profile, and international review of best practice
University of the Sunshine Coast, Queensland
Master of Science, University of the Sunshine Coast, Queensland
2026
DOI:
https://doi.org/10.25907/00998
Abstract
In certain clinical scenarios, such as multiparity or repeated whole blood transfusions, patients develop antibodies that are directed against foreign red blood cell (RBC) antigens (alloantibodies). For patients with multiple alloantibodies, or rare blood groups, it may be difficult to source antigennegative RBC to transfuse. However, not all alloantibodies will initiate damage to transfused RBC following exposure to their concordant antigens. Antibodies may be clinically significant, not clinically significant, or have variable clinical significance. The monocyte monolayer assay (MMA) is used to determine whether an alloantibody will induce donor RBC damage in vivo. However, the mechanisms underpinning the MMA are poorly understood and there is a lack of standardisation between laboratories that use it. This project aimed to investigate aspects of the MMA, including global procedures and practices, underlying mechanisms, and alternative methods for a higher throughput and more objective results. Published international MMA procedures were assessed by review and a survey, which was distributed to blood providers internationally. Two commonly used readout indexes were then compared – the phagocytic index (PI) and the monocytic index (MI). The THP-1 cell line was investigated as an alternative monocyte source. To determine the suitability of THP-1 cells in the MMA, the MI and PI readouts of peripheral blood mononuclear cells (PBMC) and THP-1 were compared. Finally, the inflammatory profiles of MMA supernatants from assays that
used plasma containing clinically significant and not clinically antibodies, as well as from THP-1 MMA, were determined by cytometric bead array, to investigate the possibility of an alternative readout and explore the underlying cellular mechanisms of the MMA. The project identified a high degree of variability in MMA methods between blood providers internationally and demonstrated inconsistency in MMA when different readout indexes were employed. THP-1 cells were shown to offer potential as an alternative monocyte source in the MMA, though they had low levels of phagocytosis. The underlying differences in MMA results between high and variable PBMC donors did not appear to be due to differences in FcγR expression, though slight differences in cellular activation status were shown. The MMA appears to involve chemokine-driven molecular processes, and the increased secretion of select chemokines offers potential for an alternative readout index.
Details
- Title
- Evaluating the monocyte monolayer assay: assay conditions, inflammatory profile, and international review of best practice
- Authors
- Isabelle Lightbody - University of the Sunshine Coast, Queensland, Centre for Bioinnovation
- Contributors
- Melinda Dean (Principal Supervisor) - University of the Sunshine Coast, Queensland, Centre for BioinnovationHelen Faddy (Co-Supervisor) - University of the Sunshine Coast, Queensland, School of Health - Biomedicine
- Awarding institution
- University of the Sunshine Coast, Queensland
- Degree awarded
- Master of Science
- Publisher
- University of the Sunshine Coast, Queensland
- DOI
- 10.25907/00998
- Organisation Unit
- School of Health
- Language
- English
- Record Identifier
- 991198151002621
- Output Type
- Thesis
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