Methodologic considerations for the use of canine in vivo aged biotinylated erythrocytes to study RBC senescence

Biotinylation of erythrocytes has been developed in rabbits as a tool to retrieve labeled cells following various periods in circulation. This retrieval capability allows biochemical studies to be conducted on red blood cells (RBC) that have aged for desired times in vivo. However, because erythrocyte life span is much shorter in rabbits than in humans, and because cell removal is measurably age-independent in rabbits, we have sought to validate the same protocol in dogs, whose cell life span and age-dependent removal characteristics are similar to humans'. Canine RBC were biotinylated in vivo by infusion of N-hydroxysuccinimidyl biotin dissolved in dimethylacetamide or dimethylsulfoxide. Cell life spans were evaluated using ¹⁴C-cyanate labeling followed by scintillation counting or avidin-FITC labeling followed by flow cytometry. Both methods gave identical results. The life span of the biotin-conjugated cells was found to be normal (~ 110 days), and the stability of the biotin ligand was adequate for efficient retrieval of cells using avidin-coated magnetic beads (magnetic cell sorting [MACS]). From each isolation, ~ 20 μL of packed biotinylated cells of ~ 90% purity (i.e., 10% contamination by unlabeled cells) could be harvested. On average, ~ 60% of the biotinylated cells in any sample could be retrieved. Either multiple isolations or use of larger collection columns will facilitate collection of cell numbers sufficient for biochemical tests. After incorporating several modifications in the previous biotinylation protocol that were required for adaptation to the dog, the methodology can be used to study red cell senescence in an animal that has several pertinent similarities to humans.