Study On The Possibility Of Shedding-relabeling Of Red Blood Cells With The Biotin Label

Objective:To discuss the reason why the red blood cell survival rate measured by biotin labeling did not decrease with time.Methods:One male C57BL/6 donor mouse was intraperitoneally injected with 100 ?L chloral hydrate.After the mouse anesthetized,blood was collected from the heart and the whole blood was promptly collected about 0.8 mL.Red blood cells(RBCs)were biotinylated in vitro,resuspended in sterile PBS after the completion of RBCs labeling to make 25% RBCs suspension.Both blood collection and biotinylation process are maintained aseptically.Based on the biotin-avidin system with high affinity,the labeled RBCs were reacted with the fluorescent streptavidin,and the labeling efficiency of the labeled RBCs were examined by flow cytometry.Then the prepared labeled RBCs(200?L per mouse)were transfused into six recipient mice by the tail vein under anesthesia.After transfusion,about 5 ?L blood samples were collected by the tail-breaking on 1 h(basal value)and 1,3,7,14,21,28 d respectively,the ratios of labeled RBCs were determined by flow cytometry.The ratio at 1 h after input was used as 100% survival of labeled RBCs in vivo and the survival rate of labeled RBCs corresponding to each time point was calculated.Time as the abscissa and survival rate as the ordinate to make the labeled RBCs survival curve plotted to further estimate posttransfusion recovery at 24 hours,half-life and mean potential life span of labeled RBCs.To further observe the shedding-relabeling phenomenon,the biotin reagent was directly injected by tail vein into other five mice for in vivo whole blood labeling.48 h after labeling,labeled RBCs were collected from mice,reacting with two different fluorescent dyes,streptavidin-R-phycoerythrin(SA-R-PE,red)and fluorescein-binding avidin(FITC-AV,green),respectively.Then the RBCs with SA-R-PE-biotin or FITC-AV-biotin(PE-RBC,FITC-RBC)were mixed and co-incubated for 4 hours at room temperature in vitro.The percentages of double-stained RBCs(PE-FITC-RBC)were detected by flow cytometry;In addition,specimens from another three mice treated with the same method and directly observed whether there are double-stained RBCs or not under confocal microscopy.Results: Posttransfusion recovery at 24 hours,half-life and mean potential life span of labeled RBCs transfused into six recipient mice was(98.9%± 15.6)%,(17.2 ± 2.4)d and(34.2 ± 4.3)d,respectively.The biotinylated RBCs decreased at a rate of(2.7 ± 0.4)% /d.However,the percentages of biotinylated RBCs in 83%(5/6)animals were higher than the baseline values.After co-incubation of PE-RBC and FITC-RBC for 4 hours in vitro,PE-FITC-RBCs were present in 5 specimens at a ratio of(0.8±0.5)% and confirmed by confocal microscopy.Conclusion:Red blood cells survival measured by biotin label are in the normal range and generally reliable;Due to the shedding-relabeling phenomenon of biotin on labeled RBCs,the survival rates of labeled RBCs transfused in vivo were not decreased but increased at some time points,but the relevant regularities need further study.