The Labile Iron Pool in Normal and Pathological Erythroid Cells - Analysis by Flow Cytometry.
Authors: Fibach Eitan , Prus Eugenia 
Published: April 2020

Abstract
The cellular "labile iron pool" (LIP) is made up of iron ions bound to low affinity ligands varying in composition and quantity under different physiological settings. It is localized primarily, but not exclusively, in the cytosol and, as such, is regarded as the crossroad of cellular iron traffic. The level of the pool is regulated and maintained within a restricted range that meets the cell requirements for iron but prevents excess from developing and triggering cellular damage. The LIP can be quantified due to its ability to bind to cell-permeable chelators, such as calcein-AM. Upon entering viable cells, calcein undergoes hydrolysis by esterases and becomes fluorescent. Its fluorescence is quenched upon binding to cellular LIP, the extent of which is correlated with the amount of LIP. The addition of a non-fluorescent, high affinity chelator, such as salicylaldehyde isonicotinoyl hydrazone (SIH), which removes the iron from the iron-calcein complex, increases the fluorescence emitted by the cells. The difference in the cellular fluorescence before and after incubation with the high affinity chelator reflects the amount of LIP. We adapted this procedure to multi-parameter flow cytometry for measuring LIP in erythroid cells derived from the peripheral blood, bone marrow and primary cultures.
The validity of the technique was determined using K562 cells - a human erythroid cell line. Cellular fluorescence increased following incubation with calcein in a concentration- and time-dependent manner. It was further augmented by cell- permeable, high affinity iron chelators such as SIH and Deferiprone (L1), but not by desferrioxamine - an impermeable chelator. Using this method, we showed that pre-incubation of the cells with iron sources such as ferrous ammonium sulfate increased their LIP level. We then studied the LIP content in peripheral blood erythroid cells. Cells were simultaneously stained with calcein and thiazol-orange, a nucleic acid specific dye, which stains reticulocytes according to their RNA content, i.e., degree of maturation. The results indicate that the LIP content decreased (69-fold) with maturation, reaching its lowest level in mature RBC. A comparison of RBC from normal donors (N=5) and patients with β-thalassemia (N=5) indicated higher a (2.4-fold) LIP in the latter. For analysis of bone marrow samples, cells were stained with calcein and fluorochrome-conjugated antibodies to surface antigens (CD45, CD71 and glycophorin A). The results indicated that the LIP content was the highest in basophilic erythroblasts and was reversely correlated with erythroid cell maturation. Finally, we studied erythroid cells in two-phase cultures of peripheral blood-derived erythroid progenitors. Following one week in the absence of erythropoietin, the cells were re-cultured in erythropoietin-supplemented medium. Analysis of the cells on different days of the second phase showed that the LIP content decreased as the cells matured and accumulated hemoglobin. The LIP content could be modulated by changing the culture conditions: increasing by supplementing normal cultures with extra iron (in the form of hollo-transferrin) and decreasing in thalassemic cultures grown in the presence of L1 or SIH.
The present findings indicate that the LIP content of erythroid cells is altered under different physiological (e.g., maturation) and pathological (e.g., iron overload, e.g., in thalassemia) conditions. The results also show that flow cytometry, a standard methodology in most hematological labs, could be useful for evaluating the LIP in various diseases and for studying the efficacy of various chelators.