Comparison of Homeostatic Reticulocyte Aging between Normal and Belgrade Rats Using Flow Cytometry.

The purpose of this work was to obtain and compare reticulocyte age distribution of normal and Belgrade (b) rats. The Belgrade rat has a microcytic, hypochromic anemia associated with abnormal reticulocyte iron uptake. Samples of blood were drawn from two homozygous rats (b/b) and three heterozygous rats (t/b). Cells were cultured over 12 hours and sampled at 3 hour intervals. The reticulocyte count was performed using a staining solution composed of thiazole orange and LDS 751, a DNA binding agent. LDS 751 was used to separate RBC form nucleated cells. The differentiation between mature RBC and reticulocytes was done by means of presence of residual RNA, which also served as a marker of reticulocyte age. The in vitro changes of the fluorescence signal of the reticulocyte population allowed us to obtain the unique relationship between the signal and the age for an individual cell based on the previously developed structured population model of cultured reticulocytes. This relation was further used to transform the in vivo signal distribution into the age distribution. The obtained reticulocyte age density was used to characterize age dependence of reticulocyte appearance in blood and death. The Belgrade rats have reticulocytosis of 13.6 ± 4% whereas control rats have reticulocytes in normal range of 2.15 ± 0.4%. The aging of reticulocyte was characterized by a relative time, RT, that a reticulocyte needs to become a mature RBC. For the normal reticulocytes the RT that an average reticulocyte spent in blood is 0.84 ± 0.02 days and ranges from 0 to 2.0 days. For Belgrade rats the time an average reticulocytes in blood needs to become a mature RBC is 1.18 ± 0.08 days and ranges from zero 0 to 2.2 days. In conclusion, the Belgrade reticulocytes are younger by about 0.4 days. The RT distributions for Belgrade rats are wider than for normal rats. Additionally, the increased death of Belgrade reticulocytes in circulation is observed. In this work the new method of studying aging of cells was successfully applied for anemic rats. It allowed us to characterize in vivo aging of normal and Belgrade reticulocytes.