Enhancement of an advanced flow cytometry method for single-cell quantification of fetal hemoglobin (HbF) and HbS in erythrocytes and reticulocytes: clinical validation and prediction of vaso-occlusive crises in Sickle Cell Disease.

• Grant number: 25/07396-4
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: September 01, 2025
• End date: February 29, 2028
• Field of knowledge: Health Sciences - Medicine - Medical Clinics
• Principal Investigator: Rodrigo Alexandre Panepucci
• Grantee: Maria Luiza Arrojo
• Host Institution: Hemocentro de Ribeirão Preto. Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da USP (HCMRP). Secretaria da Saúde (São Paulo - Estado). Ribeirão Preto , SP, Brazil

Abstract

Sickle cell disease (SCD) is caused by a mutation in the HBB gene, resulting in sickle hemoglobin (HbS), which, under hypoxia, polymerizes, deforming red blood cells and causing vaso-occlusive crises (VOCs) and severe complications. The induction of fetal hemoglobin (HbF) by hydroxyurea (HU) is an effective therapeutic strategy as it inhibits HbS polymerization. However, traditional monitoring methods, such as HPLC and classical flow cytometry (cFC), do not quantify HbF at the cellular level. HPLC measures the total percentage of HbF in the blood, while cFC only identifies the percentage of HbF+ cells (F-cells) without determining the truly protected cells. We developed a quantitative flow cytometry (qFC) method capable of determining the intracellular HbF content (cHbF) and identifying the proportion of red blood cells effectively protected (i.e., cHbF>10pg/cell). In a preliminary study (n=43), the method predicted the occurrence of VOCs in the six months following the evaluation (with 71% sensitivity and 86% specificity) for patients with less than 42% protected red blood cells. To enhance the predictive capacity of the method, we propose incorporating anti-HbS, HbA, and CD71 antibodies, enabling the quantification of protected red blood cells and reticulocytes, even in transfused patients. Two hundred SCD patients will be evaluated over the course of one year, with the collection of clinical and laboratory data, analyzing the predictive capacity of VOCs through ROC curves. The method will also be assessed for monitoring erythroid chimerism in patients undergoing hematopoietic stem cell transplants, in in vitro experiments to evaluate genetic therapies aimed at inducing HbF, such as BCL11A knockdown, and to assess the relationship between haplotypes and HbF distribution in red blood cells. The method will not only improve clinical management but also contribute to basic research on HbF regulation and the pathophysiology of SCD. (AU)