Storage-Induced Micro-Erythrocytes Can Be Quantified and Sorted by Flow Cytometry

Area of Science:

• Hematology
• Blood banking
• Cellular biology

Background:

• Refrigerated storage of red blood cell (RBC) concentrates leads to RBC alterations.
• Storage-induced micro-erythrocytes (SME), including echinocytes and spherocytes, accumulate during storage.
• SME proportion correlates with transfusion recovery and can be quantified by imaging flow cytometry (IFC).

Purpose of the Study:

• To develop a method for quantifying SME using standard flow cytometry.
• To facilitate physiologically relevant quality control of RBC concentrates.
• To enable further characterization of SME for understanding their post-transfusion fate.

Main Methods:

• RBCs stored under blood bank conditions were stained with carboxyfluorescein succinimidyl ester (CFSE).
• CFSE intensity was assessed by flow cytometry, and RBC morphology by IFC.
• Flow cytometry-based sorting was used to obtain pure SME and non-SME subpopulations.

Main Results:

• A CFSE-low RBC subpopulation, identified as SME, accumulated significantly during storage (3.3% at day 3 to 47.2% at day 42).
• Quantification of SME by flow cytometry (CFSE intensity) correlated well with IFC measurements (projected surface area).
• Flow cytometry sorting achieved ≥95% purity for CFSE-low (SME) and CFSE-high (non-SME) RBC subpopulations.

Conclusions:

• SME can be quantified using a common fluorescent dye (CFSE) and standard flow cytometry.
• This method allows for objective quality control of RBC concentrates.
• The developed staining and sorting protocol facilitates further research into SME biology and clearance.