The application of a new quantitative assay for the monitoring of integrin-associated protein CD47 on red blood cells during storage and comparison with the expression of CD47 and phosphatidylserine with flow cytometry.
BACKGROUND
After the introduction of universal leukoreduction, the role of factors other than white blood cells in red cell (RBC) storage lesion is attracting increasing attention. These include changes in the levels of CD47 and phosphatidylserine (PS) markers on RBCs during storage. The aim of this study was to monitor these changes with both flow cytometry (FACS) and a newly developed quantitative enzyme-linked immunosorbent assay (ELISA).


METHODS
A new quantitative ELISA (monoclonal antibody immobilization of RBC antigens [MAIRA]) was developed. The assay yielded consistent linear curves that enabled the measurement of CD47 expression on RBCs. In addition, FACS was used to measure both CD47 expression and PS on RBCs (n = 3 units) during storage (Days 4, 10, 24, and 31).


RESULTS
A significant reduction in CD47 expression was observed both by MAIRA assay and by FACS by Days 24 and 31 (p < 0.01), and the correlation between the two assays was significant (p < 0.01). In addition, a significant increase in PS was observed by the same storage days with FACS (p < 0.01).


CONCLUSIONS
The MAIRA assay appears to be suitable for the quantitative measurement of RBC markers during storage. Significant changes in CD47 and PS levels were observed during storage, which may have detrimental immunomodulatory and hemostatic effects on the transfused RBCs.
Stewart
Andrew
A
Immunohematology Research Group, National Science Laboratory, Scottish National Blood Transfusion Service-Edinburgh and Aberdeen, 21 Ellen's Glen Road, Edinburgh EH17 7QT, Scotland, UK.
Urbaniak
Stan
S
Turner
Marc
M
Bessos
Hagop
H
D015703:Antigens, CD / Q000032:analysis / Q000378:metabolism*; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D000094902:Blood Banking / Q000379:methods; D001793:Blood Preservation / Q000379:methods*; D051928:CD47 Antigen; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D010718:Phosphatidylserines / Q000032:analysis / Q000378:metabolism*
D015703:Antigens, CD; D015415:Biomarkers; D051928:CD47 Antigen; C497894:CD47 protein, human; D010718:Phosphatidylserines