A low-cost, accurate method for detecting reticulocytes at different maturation stages based on changes in the mitochondrial membrane potential

Abstract

In the clinical setting, reticulocytes are used as an index for the hematopoietic function of the bone marrow. Different maturation stages of reticulocytes are early markers for bone marrow hematopoietic stem cell transplantation and bone marrow regeneration after chemotherapy. Therefore, we aimed to establish a method for detecting the different reticulocyte maturation stages. Based on the decreases in mitochondrial membrane potential during reticulocyte maturation, we used MitoTracker Green (MTG)/tetramethylrhodamine, ethylester (TMRE) to identify the different reticulocyte maturation stages and used Hoechst33342 to exclude nucleated cells. The results show that this method was universal and could be applied to detect the proportions of reticulocytes in different samples. Their proportion in normal peripheral blood, a blood deficiency model, bone marrow, and spleen were (6 ± 2)%, (38 ± 4)%, (14 ± 4)%, and (3 ± 1)%, respectively. The results obtained using this method were similar to those obtained using the manual counting method (methylene blue); the correlation was good (R = 0.817; p < .01) and the coefficient of variation was lower for the method established. Moreover, reticulocytes in peripheral blood could be further divided into three distinct maturation stages: R1 (MTGneg/TMREhigh), R2 (MTGhigh/TMREhigh), and R3 (MTGhigh/TMREneg). Reticulocytes in the bone marrow and spleen could be further divided into four distinct maturation stages: R1 (MTGneg/TMREhigh), R2–1 (MTGhigh/TMREhigh/FSbig), R2–2 (MTGhigh/TMREhigh/FSsmall), and R3 (MTGhigh/TMREneg). Based on changes in mitochondrial membrane potential, MTG/TMRE/Hoechst33342 staining could be used to detect reticulocytes in different samples and at different maturation stages with low cost and high accuracy.