Isolation and analysis of residual leucocytes from leucoreduced red blood cell units

Rena Hirani; Melinda M Dean; David O Irving

doi:10.1111/vox.13775

Isolation and analysis of residual leucocytes from leucoreduced red blood cell units

Vox Sang. 2024 Nov 25. doi: 10.1111/vox.13775. Online ahead of print.

Authors

Rena Hirani^{1

2}, Melinda M Dean^{3

4}, David O Irving^{1

5}

Affiliations

¹ Australian Red Cross Lifeblood, Sydney, Australia.
² School of Natural Sciences, Macquarie University, Sydney, Australia.
³ School of Health, University of the Sunshine Coast, Sippy Downs, Queensland, Australia.
⁴ Australian Red Cross Lifeblood, Brisbane, Australia.
⁵ Faculty of Health, University of Technology Sydney, Sydney, Australia.

PMID: 39586590
DOI: 10.1111/vox.13775

Abstract

Background and objectives: Leucoreduction is used to remove donor leucocytes during red blood cell (RBC) manufacture. However, not all are removed, and long-term survival of donor leucocytes, termed transfusion-associated microchimerism (TAM), has been shown to occur in some patients following RBC transfusion. The mechanism of TAM occurrence is unknown. One hypothesis is that viable donor haematopoietic cells remain within RBC units that could engraft. However, the analysis of cells remaining within leucoreduced RBC units has been minimal. This study aimed to isolate and analyse any residual leucocytes recovered from leucoreduced RBC units.

Materials and methods: Leucoreduced RBC units were analysed on Day 1 (n = 4) and Day 42 (n = 4) post collection. Residual leucocytes were isolated using the EasySep™ RBC Depletion Reagent. Cell type analysis was conducted by flow cytometry using a leucocount reagent, a viability marker (7-amino-actinomycin D [7AAD]) and specific antibodies to CD45 and CD34. A representative 'pre-filter' sample was also obtained at the time of whole-blood donation to ensure expected cell counts across the donor samples.

Results: Analysis of the pre-filter sample showed that CD45+/CD34+ cells accounted for 0.02%-0.07% of all leucocytes. Up to 253,850 residual leucocytes were isolated across both storage timepoints, and of these, up to 48 cells were CD45+/CD34+/7AAD-.

Conclusion: Viable CD45+/CD34+ cells were isolated from leucoreduced RBC units, indicating the potential for donor progenitor cells to be present during transfusion. Further characterization of these residual cells is required to explain how TAM may occur in some patients following RBC transfusion.

Keywords: leucocytes; leucoreduction; microchimerism; red blood cell; stem cell.