Novel biomarkers for sickle cell disease and other hereditary hemolytic anemias

Abstract

The aim of this thesis was to develop and validate new biomarkers in hereditary hemolytic anemia. A new application of ektacytometry, so called oxygen gradient ektacytometry, that characterizes red cell sickling, is described in Part I of this thesis. In Chapter 2 the potential and methodological aspects of oxygen gradient ektacytometry are evaluated. Chapter 3, a video article, provides the scientific community with the exact protocol and knowledge to correctly perform oxygen gradient ektacytometry measurements. Additional factors, such as timing of the measurement, technical settings and amount of RBCs, that can influence results of this technique are reported in Chapter 4.

Part II of this thesis gives insight in how oxygen gradient ektacytometry relates to the occurrence of complications in individuals with SCD. In Chapter 5 the association of oxygen gradient ektacytometry-derived biomarkers with VOC is described, next to the response of those biomarkers to standard of care therapy. Further clinical validation of oxygen gradient ektacytometry is described in Chapter 6 in which the association with acute chest syndrome, cerebral infarction and VOC is assessed. Besides clinical validation other applications of oxygen gradient ektacytometry in SCD are explored. Chapter 7, describes a case of twins with SCD in which optimal hydroxyurea dosing is achieved with oxygen gradient ektacytometry. In Chapter 8, a new therapeutic agent mitapivat, an allosteric PK activator, is tested ex vivo in RBCs from SCD patients with oxygen gradient ektacytometry as a functional read out for efficacy. As a part of an in depth characterization of RBCs of both homozygous HbS (HbSS) and compound heterozygous for HbS and hemoglobin C (HbSC) patients, oxygen gradient ektacytometry can aid in further unraveling the pathophysiology of sickle cell disease, which is evaluated in Chapter 9.

Part III of this thesis describes additional ektacytometry methods in other hereditary hemolytic anemia’s. In Chapter 10 the effects of mitapivat are evaluated in an ex vivo study of RBCs from patients with PK deficiency. Osmotic gradient ektacytometry, amongst other outcome parameters such as PK activity and stability, was used to show efficacy of this new treatment. Chapter 11 shows the effects of splenectomy, an established treatment in hereditary spherocytosis, on RBC morphology, density and deformability. The latter was assessed with osmotic gradient ektacytometry as well as the cell membrane stability test (CMST), thereby providing the first clinical validation of this technique. These 2 ektacytometry based tests are also described in Chapter 12 which shows similarities and differences between PIEZO1 and KCNN4 mutations both resulting in hereditary xerocytosis. The findings of this thesis are summarized and put into a broader perspective in Chapter 13.