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Erythrocyte Survival as Measured by Biotin-Labelling

Normal human erythrocytes survive for 110-120 days (40-50 in mice) in circulation. In certain disease states, the survival time is decreased (Fig. 2). Some current therapies are directed at increasing the survival time of erythrocytes.

Biotin, a small water soluble B vitamin, binds readily to erythrocytes and can be used to label a subpopulation of cells (or all cells for mice) for analysis. The red blood cells (RBC) with biotin bound on the surface can be recognized by flowcytometry with a fluorescently labeled avidin molecule, which will bind with strong affinity to the biotin (Fig. 1). Each week, a blood sample is analyzed in which the bioitn-bound proportion will decrease in comparison to the whole, as new cells are generated daily (Fig. 2).

Figure 1. The biotinylated RBC are identified by flowcytometry with the use of phycoerythrin-labeled streptavidin (PESA). The unlabeled RBC have a low forward scatter (FSC-A) signal and no fluorescence (<100 on the y-scale). Any cell labeled with PESA will show a 10 to 100-fold increase in fluorescence (1,000-10,000 on the y-scale). The gate is drawn around the fluorescent events and shows the percent of the population remaining biotinylated.

Figure 2. The subjects are monitored for percent biotinylation each day to each week. Wild type mice (WT) show a 50-day RBC survival while thalassemic (B-thal) mice and mice with human sickle hemoglobin (SS) show a decreased RBC survival (35 and 15 days respectively).