Date Published: March 4, 2012
Publisher: Hindawi Publishing Corporation
Author(s): Mutaz Dana, Eugenia Prus, Eitan Fibach.
We studied the nature of enucleated RBCs containing DNA remnants, Howell-Jolly (HJ) RBCs and reticulocytes (retics), that are characteristically present in the circulation of thalassemic patients, especially after splenectomy. Using flow cytometry methodology, we measured oxidative status parameters of these cells in patients with β-thalassemia. In each patient studied, these cells had higher content of reactive oxygen species and exposed phosphatidylserine compared with their DNA-free counterparts. These results suggest that oxidative stress in thalassemic developing erythroid precursors might, through DNA-breakage, generate HJ-retics and HJ-RBCs and that oxidative stress-induced externalization of phosphatidylserine is involved in the removal of these cells from the circulation by the spleen, a mechanism similar to that of the removal of senescent RBCs.
The development of red blood cells (RBCs) from their progenitors in the bone marrow includes the process of enucleation in which the final stages of nucleated erythroid precursors (orthochromatic normoblasts) expel their nuclei to generate enucleated reticulocytes (retics), which leave the marrow and mature into RBCs . Normally, nucleated RBCs (normoblasts) are undetectable in the circulation, but in some hematological pathologies (e.g., thalassemia and sickle cell disease) they can be found in large numbers . These diseases are also characterized by mature RBCs and retics that contain DNA remnants, that are called Howell-Jolly (HJ) bodies . The frequency of these cells, which is very low, has been quantified using a flow cytometry technique [2–4].
Blood SamplesPeripheral blood (PB) samples were obtained from normal donors and splenectomized and nonsplenectomized patients with β-thalassemia intermedia and major. The samples were obtained from the counting vials after all diagnostic laboratory tests were completed. The research was approved by the Hadassah-Hebrew University Medical Centre Human Experimentation Review Board. The patients’ mutations and some relevant clinical parameters (e.g., transfusion and chelation therapy, splenectomy) were previously summarized . In polytransfused patients, blood samples were obtained before transfusion, that is, at least 3 weeks following the previous transfusion. Informed consent was obtained in all cases.
PB cells were simultaneously stained with an anti-CD71 Ab and PI, and either DCF or annexin-V. The anti-CD71 Ab marks the transferrin receptor, and PI the nucleic acid content. To evaluate the contribution of RNA (particularly in retics which contain small amounts of residual RNA) to the PI staining, PB cells were stained with PI in the presence or absence of RNase (0.4 mg/mL, Invitrogen, Carlsbad, CA). No difference was noted in the pattern of PI staining between these samples. The staining procedure identified cells as RBCs (CD71-PI-), HJ-RBCs (CD71-PI+), WBCs (CD71-PI++), retics (CD71+PI-), HJ-retics (CD71+PI+), and normoblasts (CD71+PI++). Figure 1(a) shows a flow cytometry dot-plot (PI versus CD71) analysis of a blood sample derived from a representative splenectomized β-thalassemic patient, indicating the various cell populations. The fluorescence distribution histograms of each cell population with respect to DCF-fluorescence, indicating generation of ROS, and annexin V-fluorescence, indicating exposed PS, with their MFIs, are shown in Figures 1(b) and 1(c), respectively. The results indicate higher ROS and PS in retics than in mature RBCs, and, more critically, in HJ-cells compared with their non-HJ counterparts: in the experiment presented in Figure 1(b), showing ROS results, the MFI of HJ-RBCs was 2.3-fold higher than that of RBCs, and the MFI of HJ-retics was 2.4-fold higher than retics. In Figure 1(c), showing PS results, the MFI of HJ-RBCs was 15.3-fold higher than that of RBCs, and the MFI of HJ-retics was 12.1-fold higher than retics.