Differences in oxygen-gradient ektacytometry parameters and blood viscosity between patients with sickle cell anemia and patients with sickle cell-hemoglobin C disorder

Abstract

Oxygen-gradient ektacytometry (oxygenscan) has been recently used in the context of sickle cell disease (SCD) to determine the ability of red blood cells (RBCs) to deform in normoxia and during deoxygenation. While several studies focused on the determinants of oxygenscan parameters in the homozygous form of SCD (i.e., HbSS), very few studies focused on hemoglobin SC disease (HbSC). Oxygenscan parameters were compared between HbSS (N = 304) and HbSC (N = 58) patients, with or without hydroxyurea (HU). RBC deformability measured in normoxia or at low oxygen level was lower in HbSS patients without HU compared to HbSS patients with HU and HbSC patients with or without HU. The propensity of RBCs to sickle during deoxygenation was greater in HbSS than in HbSC patients. Although no significant impact of HU was observed on RBC deformability/sickling in HbSC patients, those with HU had lower blood viscosity. The ability of RBC to recover their maximum deformability after a deoxygenation/reoxygenation cycle was lower in HbSC compared to HbSS patients. Finally, oxygenscan parameters and routine hematological parameters correlated differently in HbSS and HbSC patients. Our study showed difference in oxygenscan parameters between HbSS and HbSC individuals that could partly explain the difference in the clinical expression of SCD.

Keywords

sickle cell disease red blood cell deformability sickling oxygen gradient ektacytometry

Introduction

Oxygen-gradient ektacytometry (oxygenscan) has been recently used in the context of sickle cell disease (SCD) to determine the ability of red blood cells (RBCs) to deform in normoxia and during deoxygenation. It allows the determination of maximal RBC deformability in normoxia (EImax), minimum RBC deformability (EImin) reached at low oxygen partial pressure (pO₂) and the propensity of RBCs to sickle during deoxygenation (i.e., the pO₂ at which RBCs start to sickle, called the Point of Sickling (PoS)). Although the clinical interest of these biomarkers is still under debate,^1,2 several studies reported associations with clinical severity in patients with the homozygous form of SCD (i.e., sickle cell anemia or HbSS).^3–6

Oxygenscan parameters have also been measured to investigate the effects of various treatments. Since exchange transfusion decreases the relative amount of RBCs containing abnormal hemoglobin and increases the relative amount of normal RBCs into the blood circulation, this treatment results in a decrease of PoS and an increase of both EImin and EImax in HbSS patients.^4,6 Hydroxyurea (HU), a drug that promotes the production of fetal hemoglobin (HbF), has also been reported to affect oxygenscan parameters with PoS decreasing and EImin and EImax increasing 3 months after the beginning of treatment in HbSS patients.^6,7 Chonat et al.⁸ investigated the effects of voxelotor on RBC rheology in HbSS patients and observed a decrease of PoS and an increase of EImin and EImax after 48 weeks of treatment, which led to a reduction of hemolysis and a rise in hemoglobin concentration. The effects of pyruvate kinase activators on RBCs have also been investigated both in-vivo and in-vitro. It was shown that these molecules, by increasing adenosine triphosphate (ATP) and decreasing 2,3-diphosphoglycerate levels, decreased PoS and increased EImin and EImax in HbSS individuals.^9–12

Very few studies investigated oxygenscan parameters in patients with sickle cell-hemoglobin C disorder (HbSC), another frequent form of SCD. Patients with HbSC have milder anemia than HbSS individuals and are characterized by higher blood viscosity, which contributes to the occurrence of several clinical complications.^13,14 The presence of both HbS and HbC into RBCs affect their rheological properties, with deformability being reduced compared to normal RBCs.¹⁵ Boisson et al.⁴ compared small groups of HbSS and HbSC individuals and found higher PoS and lower EImin and EImax in the former group. Joly et al.¹⁶ recently reported that HbS percentage and mean corpuscular hemoglobin concentration (MCHC) are key regulators of the PoS in HbSS individuals but no study focused on the determinants of PoS, as well as of the other oxygenscan parameters, in HbSC patients. The aim of the present study was to compare oxygenscan parameters between HbSS and HbSC patients and to test the associations with several routine hematological parameters. Since HU may impact on HbF, mean cell volume (MCV) and MCHC, patients treated with HU were separated from those without HU in the statistical comparisons.

Material and methods

Participants

A group of 362 patients with SCD (304 HbSS: 27.4 ± 13.6 yrs, 152 males, 152 females, 222 treated by HU; 58 HbSC: 34.2 ± 17.3 yrs, 26 males, 32 females, 15 treated by HU) followed by the Sickle Cell Centre of Lyon were included in the present study. All patients were at steady-state: no transfusion and no acute complication in the last 3 months. Patients under HU treatment received their medication since at least 3 months. Blood was sampled in EDTA tubes to perform hematological and hemorheological measurements. The study was conducted in accordance with the guidelines set by the Declaration of Helsinki and was approved by the Regional Ethics Committees (L14-127).

Hematological and oxygenscan parameters

Hemoglobin concentration (Hb), hematocrit (Hct), MCV and MCHC were determined with a hematology analyzer (Advia, Siemens, Rungis, France). HbS, HbC and HbF levels were measured by capillary electrophoresis (Capillarys 2 FP, Sebia, Lisses, France). Blood viscosity was measured at 225 s⁻¹ with a Brookfield DVT cone-plate viscometer and a CPE-40 cone, as previously described.¹⁷ Ektacytometry was carried out with the laser-assisted optical rotational red cell analyzer (Lorrca, RR Mechatronics, The Netherlands) with the oxygenscan module to measure RBC deformability over an oxygen gradient. A volume of 50 µL of blood, standardized to a fixed RBC count of 200 × 10⁶, was mixed with 5 mL of high viscous (30 cP) Oxy-Iso polyvinylpyrrolidone (PVP) suspension.¹⁸ The suspension was sheared at 30 Pa and 37°C into the Couette system made of glass of the ektacytometer. The oxygen partial pressure (pO₂) was gradually decreased from 160 mmHg to 20 mmHg (deoxygenation) and then re-increased to normoxic values.^7,18,19 The diffraction pattern was analyzed by the computer, and an elongation index (EI) that reflects RBC deformability was calculated. Several parameters were derived: (1) EImax, the RBC deformability at normoxia; (2) EImin, the lowest RBC deformability reached upon deoxygenation; (3) point of sickling (PoS), the pO₂ at which RBC deformability decreases below 5% of EImax during deoxygenation; (4) Delta-EI, the difference between EImin and EImax and 5) the percentage of recovery, which corresponds to the difference between EImax and the EI value reached after the end of the reoxygenation process. All measurements were standardized as recommended.¹⁸ Since blood storage (24 h or longer) may affect oxygenscan parameters,^18,19 all measurements were carried out within 4 h after sampling.

Statistics

Comparisons between the four groups (HbSS patients with HU, HbSS patients without HU, HbSC patients with HU, HbSC patients without HU) were performed using a one-way ANOVA followed by Tukey post-hoc tests when appropriate. A Pearson test was used to test the associations between oxygenscan parameters and the other parameters for HbSS and HbSC patients separately. A p value less than 0.05 was considered as statistically significant. GraphPad Prism (version 8.3) was used for the statistical analyses.

Results

Comparisons of the four groups

Figure 1 shows the results of hematological, blood viscosity and oxygenscan parameters in the four groups. HbF level was higher in HbSS compared to HbSC individuals. HU-treated patients had higher HbF than untreated patients in the HbSS genotype but not in the HbSC genotype (p ranging from < 0.01 to < 0.0001). Hb was lower in HbSS without HU compared to the 3 other groups (p < 0.0001). HbSS with HU had lower Hb than HbSC with (p < 0.001) and without HU (p < 0.0001). No significant difference in Hb was observed between HbSC with or without HU. Blood viscosity was not different between HbSS patients with HU and those without. HbSC individuals with and without HU had greater blood viscosity than HbSS patients without HU (p < 0.05 and p < 0.0001, respectively). HbSC patients with HU had lower blood viscosity than HbSC patients without (p < 0.05). EImin of HbSS patients without HU was lower compared to HbSC individuals with and without HU (p < 0.05). HbSC patients with HU had higher EI min than HbSS patients with HU (p < 0.05). No difference was observed between the two HbSC groups for EImin. EImax of HbSS patients without HU was lower compared to HbSS patients with HU (p < 0.0001), HbSC with HU (p < 0.01) and HbSC patients without HU (p < 0.05). No other difference was observed for EImax. PoS was lower in the two HbSC groups (p < 0.05 and p < 0.001) compared to HbSS patients without HU. HbSC patients without HU had lower PoS than HbSS patients with HU (p < 0.05). No PoS difference was observed between HbSS with and without HU, or between HbSC patients with and without HU. Delta-EI of HbSS patients with HU was higher compared to HbSS patients without HU (p < 0.01) and HbSC patients without HU (p < 0.001). No other difference was observed for Delta-EI. The percentage of recovery of EI was systematically lower in HbSC compared to HbSS patients (p ranging from < 0.01 to < 0.0001). No difference in the percentage of recovery was observed between the two HbSC groups while we observed slightly lower values in HbSS patients with HU compared to those without (p < 0.01). MCV of HbSS individuals with HU was higher compared to those without (p < 0.0001) and to the two HbSC groups (p < 0.0001). MCV was not different between the two HbSC groups. MCHC of HbSS patients without HU was lower compared to the three other groups (p ranging between < 0.05 and < 0.0001). HbSS patients with HU had lower MCHC than HbSC patients without HU (p < 0.01). No difference in MCHC was observed between HbSC patients with and without HU.

Figure 1.

Comparisons of blood viscosity and of the oxygenscan and hematological parameters between HbSS patients with HU, HbSS patients without HU, HbSC patients with HU and HbSC patients without HU. HbF = Fetal hemoglobin, Hb = hemoglobin, EImin = minimum RBC deformability reached at low oxygen partial pressure (pO₂), EImax = maximal RBC deformability in normoxia, PoS = point of sickling (i.e., the pO₂ at which RBCs start to sickle), Delta-EI = difference between EImin and EImax, recovery = difference between EImax and the EI value reached after the end of the reoxygenation process, MCV = mean cell volume, MCHC = mean corpuscular hemoglobin concentration. Significant difference: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Correlations with oxygenscan parameters

Figure 2 shows the correlation matrix (r values) between all parameters for HbSC and HbSS individuals.

Figure 2.

Correlations matrix (r values are indicated) between oxygen gradient ektacytometry and hematological parameters in HbSS (SS) and HbSC (SC) patients. Colored squares indicate the level of statistical significance.

For HbSS patients: 1) EImin did not correlate with MCHC (p = 0.59), positively correlated with HbF (p < 0.0001) and MCV (p < 0.0001), and negatively with HbS (p < 0.0001); 2) EImax positively correlated with HbF (p < 0.0001) and MCV (p < 0.0001), and negatively with HbS (p < 0.0001) and MCHC (p < 0.01); 3) PoS negatively correlated with HbF (p < 0.0001) and MCV (p < 0.05) and positively with HbS (p < 0.0001) and MCHC (p < 0.05); 4) Delta-EI did not correlate with HbF (p = 0.39), HbS (p = 0.51) or MCV (p = 0.70) but correlated negatively with MCHC (p < 0.05); 5) The percentage of recovery negatively correlated with MCHC (p < 0.05) but did not correlate with HbF (0.56), HbS (p = 0.26) or MCV (p = 0.74).

For HbSC patients: 1) EImin was negatively correlated with HbS (p < 0.05) and HbC (p < 0.05), and positively with HbF (p < 0.05) but no correlation was observed with MCV (p = 0.29) or MCHC (p = 0.44); 2) EImax was negatively correlated with MCHC (p < 0.05) but no correlation was found with the other parameters (HbF: p = 0.85; HbS: p = 0.94; HbC: p = 0.96; MCV: p = 0.58); 3) no correlation between PoS and the other parameters was observed (HbF: p = 0.96; HbS: p = 0.81; HbC: p : 0.69; MCV: p = 0.23; MCHC: p = 0.61); 4) Delta-EI negatively correlated with HbF (p < 0.05) and positively with HbS and HbC (p < 0.05), but no correlation was observed with MCV (p = 0.15) or MCHC (p = 0.24); 5) only MCV positively correlated with the percentage of recovery (p < 0.01; HbF: p = 0.70; HbS: p = 0.83; HbC: p = 0.28; MCHC: p = 0.52).

Discussion

The present study mainly showed that 1) RBC deformability measured in normoxia or at low oxygen level was lower in HbSS patients without HU compared to the three other groups, 2) the propensity of RBC to sickle during deoxygenation was greater in HbSS than in HbSC patients, 3) although no significant impact of HU was observed on Hb and RBC deformability/sickling in HbSC patients, those with HU had lower blood viscosity, 4) the percentage of RBC deformability recovery during reoxygenation was lower in HbSC compared to HbSS patients, 5) oxygenscan parameters correlated differently with HbS, HbC, HbF, MCHC or MCV in HbSS and HbSC patients.

As shown in our study, HbSC patients are usually marked by milder anemia than HbSS patients, leading to chronic blood hyperviscosity, which plays a major role in the occurrence of chronic organ complications such as retinopathy, otologic disorders or osteonecrosis.^13,14,20 Although no difference in Hb level was observed between the two HbSC groups, patients with HU exhibited a lower blood viscosity. This difference could be attributed to the slightly higher, although not significant, RBC deformability measured in normoxia and at low oxygen level in HU-treated HbSC patients compared to the untreated ones. Luchtman-Jones et al.²¹ reported increased HbF level and MCV in HbSC patients treated with HU. Although no significant difference in HbF level, MCV or MCHC was observed between the two HbSC subgroups, the correlation analyses suggested that HbS and HbF levels, and MCHC may modulate RBC deformability in HbSC individuals. Indeed, drugs targeting HbF, MCV and/or MCHC in HbSC individuals could result in greater RBC deformability, which in turn could lower blood viscosity for a given Hb level, as it is the case in HbSS patients treated by HU.²² The lowering of blood viscosity in HbSC patients could be expected to decrease the risk for vaso-occlusive like events as it does in HbSS individuals.^23–25 Beside, Luchtman-Jones et al.²¹ previously observed a reduction of painful events in HbSC patients older than 15 yrs old treated by HU.

When blood was deoxygenated, the propensity of RBCs to sickle (i.e., PoS) was higher in HbSS individuals compared to HbSC patients, leading RBC deformability to reach lower values in HbSS patients (i.e., EImin). EImax, EImin and PoS were associated to MCV, MCHC, HbF and HbS in HbSS patients, which may explain why patients treated by HU are usually characterized by higher EImax and EImin, and lower PoS.^4,6 The present study only found higher EImax in HbSS patients treated by HU compared to those without, but the cross-sectional nature of the study does not allow to really investigate the impact of HU on EImin and PoS. It was recently proposed that oxygen-gradient ektacytometry could be useful for dose titration of hydroxyurea therapy in children with HbSS.²⁶ While EImin of HbSC patients was associated with HbS, HbC and HbF, PoS was not associated with any of the parameters investigated. It is thus unknown if HU treatment in HbSC patients would decrease the PoS but one could suspect that it would improve EImin. The ongoing clinical trial focusing on the longitudinal effects of HU in HbSC patients, which incorporated oxygen gradient ektacytometry measurements, should help in providing a clear answer on these points.²⁷

One of the most surprising findings of the present study is the lowest ability of RBCs from HbSC individuals compared to HbSS patients to recover their normoxic deformability after a full cycle of deoxygenation. The percentage of recovery from HbSC individuals was positively correlated with MCV, suggesting that the hydration status of RBCs could play a key role in the ability of RBCs to recover their normoxic deformability after a deoxygenation cycle. This finding might be particularly important in the context of HbSC since it has been demonstrated that RBC tend to be very dehydrated because of the increased K:Cl cotransporter (KCC) activity in this sickle cell genotype.²⁸ Whether the percentage of recovery would further decrease after several oxygenation/deoxygenation cycles is unknown and further studies are needed to test the clinical usefulness, if any, of this parameter in the context of SCD.

Although, the clinical usefulness of oxygen-gradient ektacytometry in SCD has been questioned,¹ several studies found some associations between oxygen-gradient ektacytometry parameters and several clinical complications. For instance, Rab et al.⁶ reported that the PoS was higher and EImin was lower in both HbSS adults and children with frequent vaso-occlusive crises compared to those who did not experience any vaso-occlusive crises in the 2 yrs preceding the inclusion in the study. This finding has been reinforced by another study⁵ where it was demonstrated that for every 10 mmHg increase in PoS, the likelihood of an HbSS individual experiencing >1 type of acute complication increased (adjusted odds ratio = 1.65). In another study, Boisson et al.⁴ found that the PoS was increased in HbSS individuals during vaso-occlusive crises compared to steady-state, suggesting a greater propensity of RBCs to sickle upon deoxygenation during this acute complication. However, it must be acknowledged that, at that time, there is no proof of the superiority of the oxygen-gradient ektacytometry parameters, compared to more well-known biomarkers of SCD such as HbF, F-cells, and dense RBCs,²⁹ or compared to shear-stress gradient ektacytometry parameters,³ in predicting the onset of a clinical complication. However, oxygen-gradient ektacytometry seems to be very useful for testing the effects of some medications/new molecules targeting HbS polymerization/RBC sickling or to identify new mechanisms that could modulate RBC sickling.^{8,9,12,30–35}

In conclusion, our study showed difference in oxygenscan parameters and blood viscosity between HbSS and HbSC individuals that could partly explain the differences in the clinical and hematological expression of the disease. Future studies focusing on the effects of various drugs in HbSC patients should account for the distinct determinants of RBC rheology and sickling in this genotype, which may influence treatment responses.

Footnotes

Author contribution

PC, MM, CB, ES, CR, PJ, and EN performed the experiments. SP, CHD and AG recruited the patients. PC, CB and EN designed the research study. PC and EN analyzed the data. PC and EN wrote the paper. All authors carefully read the paper and approved the final version.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

ORCID iD

Philippe Connes

References

Nardo-Marino

Petersen

Brewin

, et al. Oxygen gradient ektacytometry does not predict pain in children with sickle cell anaemia. Br J Haematol 2022; 197: 609–617.

Sheehan

van Beers

Connes

, et al. Comment on: oxygen gradient ektacytometry does not predict pain in children with sickle cell anaemia. Br J Haematol 2022; 197: e61–e62.

Boisson

Nader

Renoux

, et al. Shear-stress-gradient and oxygen-gradient ektacytometry in sickle cell patients at steady state and during vaso-occlusive crises. Cells 2022; 11: 585.

Boisson

Rab

MAE

Nader

, et al. Effects of genotypes and treatment on oxygenscan parameters in sickle cell disease. Cells 2021; 10: 811.

Rab

MAE

Kanne

Boisson

, et al. Oxygen gradient ektacytometry-derived biomarkers are associated with acute complications in sickle cell disease. Blood Adv 2024; 8: 276–286.

Rab

MAE

Kanne

Bos

, et al. Oxygen gradient ektacytometry-derived biomarkers are associated with vaso-occlusive crises and correlate with treatment response in sickle cell disease. Am J Hematol 2021; 96: E29–E32.

Rab

MAE

van Oirschot

Bos

, et al. Rapid and reproducible characterization of sickling during automated deoxygenation in sickle cell disease patients. Am J Hematol 2019; 94: 575–584.

Chonat

Fields

Baratz

, et al. Voxelotor improves red blood cell functionality in children with sickle cell anaemia: an ancillary study of the HOPE-KIDS 1 trial. EJHaem 2024; 5: 125–130.

Joly

Nader

Ketels

, et al. Effects of pyruvate kinase activators on red blood cell properties in sickle cell disease. Br J Haematol 2023; 202: e27–e30.

10.

Rab

MAE

Van Oirschot

Kosinski

, et al. AG-348 (Mitapivat), an allosteric activator of red blood cell pyruvate kinase, increases enzymatic activity, protein stability, and ATP levels over a broad range of PKLR genotypes. Haematologica 2021; 106: 238–249.

11.

Schroeder

Fulzele

Forsyth

, et al. Etavopivat, a pyruvate kinase activator in red blood cells, for the treatment of sickle cell disease. J Pharmacol Exp Ther 2022; 380: 210–219.

12.

van Dijk

Rab

MAE

van Oirschot

, et al. One-year safety and efficacy of mitapivat in sickle cell disease: follow-up results of a phase 2, open-label study. Blood Adv 2023; 7: 7539–7550.

13.

Lemonne

Lamarre

Romana

, et al. Impaired blood rheology plays a role in the chronic disorders associated with sickle cell-hemoglobin C disease. Haematologica 2014; 99: 74–75.

14.

Lemonne

Romana

Lamarre

, et al. Association between relative systemic hypertension and otologic disorders in patients with sickle cell-hemoglobin C disorder. Am J Hematol 2014; 89: 667.

15.

Tripette

Alexy

Hardy-Dessources

, et al. Red blood cell aggregation, aggregate strength and oxygen transport potential of blood are abnormal in both homozygous sickle cell anemia and sickle-hemoglobin C disease. Haematologica 2009; 94: 1060–1065.

16.

Joly

Boisson

Renoux

, et al. Determinants of the point of sickling measured by oxygen gradient ektacytometry in sickle cell anaemia. Br J Haematol 2022; 197: e56–e58.

17.

Baskurt

Boynard

Cokelet

, et al. New guidelines for hemorheological laboratory techniques. Clin Hemorheol Microcirc 2009; 42: 75–97.

18.

Rab

MAE

Kanne

Bos

, et al. Methodological aspects of the oxygenscan in sickle cell disease: a need for standardization. Am J Hematol 2020; 95: E5–E8.

19.

Boisson

Rab

MAE

Nader

, et al. Methodological aspects of oxygen gradient ektacytometry in sickle cell disease: effects of sample storage on outcome parameters in distinct patient subgroups. Clin Hemorheol Microcirc 2021; 77: 391–394.

20.

Lionnet

Hammoudi

Stojanovic

, et al. Hemoglobin sickle cell disease complications: a clinical study of 179 cases. Haematologica 2012; 97: 1136–1141.

21.

Luchtman-Jones

Pressel

Hilliard

, et al. Effects of hydroxyurea treatment for patients with hemoglobin SC disease. Am J Hematol 2016; 91: 238–242.

22.

Lemonne

Charlot

Waltz

, et al. Hydroxyurea treatment does not increase blood viscosity and improves red blood cell rheology in sickle cell anemia. Haematologica 2015; 100: e383–e386.

23.

Charlot

Romana

Moeckesch

, et al.

Which side of the balance determines the frequency of vaso-occlusive crises in children with sickle cell anemia: blood viscosity or microvascular dysfunction?

Blood Cells Mol Dis 2016; 56: 41–45.

24.

Lamarre

Romana

Waltz

, et al. Hemorheological risk factors of acute chest syndrome and painful vaso-occlusive crisis in children with sickle cell disease. Haematologica 2012; 97: 1641–1647.

25.

Nebor

Bowers

Hardy-Dessources

, et al. Frequency of pain crises in sickle cell anemia and its relationship with the sympatho-vagal balance, blood viscosity and inflammation. Haematologica 2011; 96: 1589–1594.

26.

Houwing

Bos

van Wijk

, et al. Use of the oxygen gradient ektacytometry in the dose titration of hydroxyurea therapy in children with sickle cell disease. Int J Lab Hematol 2024; 46: 390–394.

27.

Segbefia

Smart

Stuber

, et al. Baseline characteristics of Ghanaian children and adults enrolled in PIVOT, a randomised clinical trial of hydroxyurea in HbSC disease in sub-Saharan Africa. Br J Haematol 2024; 205: 2470–2480.

28.

Hannemann

Rees

Tewari

, et al. Cation homeostasis in red cells from patients with sickle cell disease heterologous for HbS and HbC (HbSC genotype). EBioMedicine 2015; 2: 1669–1676.

29.

Sadaf

Seu

Thaman

, et al. Automated oxygen gradient ektacytometry: a novel biomarker in sickle cell Anemia. Front Physiol 2021; 12: 636609.

30.

Hatem

Esperti

Murciano

, et al. Adverse effects of delta-9-tetrahydrocannabinol on sickle red blood cells. Am J Hematol 2023; 98: E383–E386.

31.

Nader

Conran

Leonardo

, et al. Piezo1 activation augments sickling propensity and the adhesive properties of sickle red blood cells in a calcium-dependent manner. Br J Haematol 2023; 202: 657–668.

32.

Bordat

Connes

Joly

, et al. Impact of elevated red blood cell membrane cholesterol in sickle cell anemia patients: effects of BRN-002, a 2-hydroxypropyl-beta-cyclodextrin derivate, on red blood cell lipids, deformability, sickling and hemolysis. Blood Cells Mol Dis 2025; 113-114: 102939.

33.

van Dijk

Rab

MAE

van Oirschot

, et al. Safety and efficacy of mitapivat, an oral pyruvate kinase activator, in sickle cell disease: a phase 2, open-label study. Am J Hematol 2022; 97: E226–E229.

34.

Bordat

Nader

Connes

, et al. Effect of dalcetrapib, voxelotor and their combination on red blood cell deformability and sickling in sickle cell disease. Clin Hemorheol Microcirc 2025; 89: 335–339.

35.

Saraf

Hagar

Idowu

, et al. Multicenter, phase 1 study of etavopivat (FT-4202) treatment for up to 12 weeks in patients with sickle cell disease. Blood Adv 2024; 8: 4459–4475.