Due to the challenges associated with accessing bone marrow samples from individuals with sickle cell anemia (SCA), hematopoiesis in the context of this disease remains less studied compared to other pathologies. However, characterizing the circulating hematopoietic stem and progenitor cell (HSPC) populations is feasible and may provide insights into the hematopoietic phenotype in patients. Moreover, the characterization of HSPCs in SCA, especially among patients under different treatment protocols, holds significant value from the perspective of gene therapy. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors (n = 9) and patients with SCA exhibiting high levels of circulating erythroblasts (n = 9). Briefly, PBMCs were obtained through density gradient media centrifugation. Subsequently, 1 million cells per sample were stained with an antibody panel targeting HSPC markers (anti-human CD10, CD34, CD38, CD45, CD45RA, CD90, and CD123) for the identification of various hematopoietic cell populations, including hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), megakaryocyte/erythrocyte progenitors (MEPs), granulocyte/macrophage progenitors (GMPs), and common lymphoid and myeloid progenitors (CLPs and CMPs), as well as erythroblasts (CD34, CD71, CD105, CD117, CD123, CD235a) including burst-forming unit erythroid and colony-forming unit erythroid (BFU-e and CFU-e), proerythroblasts, early and late basophilic erythroblasts, polychromatic erythroblasts, and orthochromatic erythroblasts. After incubation, samples were acquired using a CytoFLEX S flow cytometer (Beckman Coulter), and the resulting files were further analyzed with FlowJo software (BD Biosciences). The computed data were analyzed to statistically compare mean scores using Kruskal-Wallis with Dunn's multiple comparison tests. Significance levels are indicated as < 0.05, where ’*’, ’**’, ’, and ’correspond to values of ≤ 0.05, 0.01, 0.001, and 0.0001, respectively. Overall, SCA samples exhibited higher counts of HSCs (****), MPPs (**), CMPs (***), and GMPs (**). In contrast, CLPs (**) were more prevalent in donor samples. While the overall count of CD34+ cells was higher among SCA samples (**), these samples demonstrated a contrasting median fluorescence intensity (MFI) (****), which was higher among donors. Concerning circulating erythroid progenitors, SCA samples also showed higher numbers of BFU-e and CFU-e (***). This trend was also observed for basophilic, polychromatic, and orthochromatic erythroblasts (**). These results suggest an abnormal phenotype for circulating HSPCs and erythroblasts in SCA. It remains unclear whether this reflects a compensatory mechanism for the recognized ineffective hematopoiesis and stress erythropoiesis inherent to SCA or a pathology-induced alteration in the function of these cell compartments. To address this question, our subsequent approaches will focus on elucidating whether the function of these cells is intact and how the most prominent pathological features in SCA, such as systemic inflammation and oxidative stress, impact hematopoiesis and erythropoiesis.

FUNDHERP, CTC/FAPESP (2013/08135-2), INCTC/CNPq (465539/2014-9), CAPES.