New Insights in Sickle Cell Disease Provided By Intravascular Hemolysis and Total Hb Mass Parameters
Authors: De Luna Gonzalo , Habibi Anoosha , Le Corvoiser Philippe , Martino Suella , Alhamrouni Jamila , d'Humières Thomas , Audureau Etienne , Natella PA , Bencheikh Laura , Perkins Meghan , Cambot Marie , Frederic Galacteros , Bartolucci Pablo 
Published: December 2023

New biomarkers may provide a better understanding of the complex pathophysiology of sickle cell disease. Anemia is defined as a reduction in the blood concentration of hemoglobin ([Hb]) to &lt; 12 g/dL in females and &lt; 13 g/dL in males. [Hb] will depend not just on the total circulating quantity of hemoglobin (tHbmass), but also on plasma volume (PV) in which it is suspended. Exercise capacity correlates poorly with [Hb], in contrast to tHbmass, as shown by studies comparing elite athletes and sedentary individuals. Similarly, classic parameters of hemolysis (LDH, bilirubin, ASAT) are not very specific for erythrocytes and do not always reflect the intravascular component of hemolysis. Plasma hemoglobin and heme, released by hemoglobin oxidation, are specific biomarkers of RBC destruction in the circulation and have their own deleterious effects on vascular walls, NO bioavailability, and the inflammatory state. In this study, we aimed to analyze the contribution of these biomarkers to cardiac and vascular parameters.

Method: We carried out a preliminary analysis of patients included in the HEMOPROVE trial (NCT05199766) a prospective open-label study designed to better understand the effects of Voxelotor. Patients had to be more than one month from a vaso-occlusive crisis, 3 months from a transfusion and on a stable dose of hydroxyurea (HU) for at least 3 months to be included. We extracted and analyzed data from patients who had a pre-treatment Hb mass measurement. Hb mass, plasma volume were determined using the validated oCOR method using medical device Detalo Clinical TM - CO Rebreather (IEC 60601-1 and IEC 62304 for medical devices). Only patients with a HbCO of less than 3% benefited from this measurement; plasma hemoglobin and plasma heme were measured using the technique developed by our team (BET 18P3417). Blood pressure and heart rate were measured during echocardiography. All values were collected within the same week of inclusion in the protocol. Correlations were performed using a Spearman test, paired data comparisons were made with a Wilcoxon test (Graphpad9, PRISM).

Results: Fourteen patients were included for blood mass measurement. Median age was 38 years [34.7-50.7], sex ratio F/M was 7/7, 10/14 patients were treated with HU. Median [Hb] was 7.4g/dL [6.6-7.7], tHbmass was 522 g [352-704] corresponding to 8.1 g/kg [6.1-11.7]. [Hb] and tHbmass were strongly correlated (p=0.008, r=0.68), whereas it was weaker with tHbmass/kg (p=0.046, r=0.54). Interestingly, some patients with approximately the same [Hb] had widely varying tHbmass. These results can be explained by significant variability in plasma volume, which was increased in half the patients, with a median increase of +135% [100-215] and a median volume of 5656 ml [4140-6931]. The tHbMass (p=0.01; r=0.64), tHbMass/kg (p=0.05, r=0.52) and above all PV (p=0.004; r=0.73) correlated with the E/E' used to evaluate the left ventricle filing pressure, while there was no correlation with [Hb] (p=0.5). Interestingly, the cardiac index was inversely correlated with plasma Hemoglobin (p=0.003, r=-0.74) with no correlation with [Hb] (p=0.4) or LDH (p=0.90), while mean and diastolic blood pressure were negatively correlated with plasma Heme (respectively p=0.01; r=-0.65 and p=0.001; r=-0.77) with no correlation with [Hb] and LDH (p&gt;0.4).

Conclusions: Measurements of total hemoglobin mass and intravascular hemolysis parameters provide a better understanding of the diversity of manifestations that lie behind sickle cell anemia, underpinned by complex mechanisms.