Probing Red Blood Cell Deformability in Sickle Cell Disease, Sickle Cell Trait, and Healthy Controls by Ektacytometry

ABSTRACT

Introduction

Red blood cell (RBC) deformability is a key determinant of sickle cell disease (SCD) pathophysiology. We evaluated RBC deformability across HbSS, HbAS, and HbAA genotypes and investigated its associations with hemolysis, fetal hemoglobin (HbF), coinherited α‐thalassemia, hydroxyurea treatment, and the PIEZO1 E756del variant.

Methods

We used Lorrca ektacytometry to compare RBC deformability across 174 participants with HbSS, HbAS, and HbAA genotypes. Deformability was evaluated across a range of shear stresses, osmotic pressures, and oxygen tensions. Associations between deformability and hemolysis markers, HbF levels, α‐thalassemia status, hydroxyurea use, and the PIEZO1 E756del variant were assessed.

Results

HbSS RBCs showed markedly reduced deformability across all shear stresses, osmotic pressures, and oxygen tensions compared to HbAS and HbAA RBCs. Reduced deformability correlated with elevated markers of hemolysis, including lactate dehydrogenase (LDH) and bilirubin. Higher HbF levels were independently associated with improved RBC deformability, whereas coinherited α‐thalassemia was not associated with improved deformability. Hydroxyurea use was associated with significantly improved deformability. The PIEZO1 E756del variant was associated with reduced deformability among participants with HbSS.

Conclusion

RBC deformability is substantially impaired in HbSS and is associated with markers of hemolysis and several genetic and treatment‐related factors. These findings support Lorrca ektacytometry as a robust biomarker platform for assessing RBC health and treatment response in SCD.

Trial Registration

ClinicalTrials.gov identifier: NCT03685721

1. Introduction

Red blood cell (RBC) deformability is the ability of RBCs to change shape under flow, enabling efficient movement through the microcirculation [1]. In sickle cell disease (SCD), a HBB gene mutation leads to the production of hemoglobin S (HbS). Under low oxygen conditions, HbS polymerizes [2], making RBCs rigid, less deformable, more adhesive, and fragile. This sickling process underlies the hallmarks of SCD: Recurrent microvascular occlusion, pain crises, chronic hemolytic anemia, organ damage, and early death [3, 4].

While reduced RBC deformability is a well‐established feature of SCD, the relative contributions of fetal hemoglobin (HbF), coinherited α‐thalassemia, hydroxyurea (HU) therapy, and genetic modifiers such as PIEZO1 variants to this phenotype remain incompletely resolved, and existing data are sometimes contradictory across cohorts. We used a single well‐characterized platform (Lorrca ektacytometry) across three hemoglobin genotypes—HbSS, HbAS (sickle cell trait), and HbAA (normal)—to directly compare deformability profiles and test associations with hemolytic markers and known genetic modifiers.

2. Methods

We enrolled 174 participants from clinical trial NCT03685721, a study approved by the National Heart, Lung, and Blood Institute (NHLBI) IRB. All participants were 18 years or older and did not receive blood transfusion for at least 3 months prior to enrollment. Participants included 72 with HbSS (48 were on HU therapy), 68 with HbAS, and 34 with HbAA. Of the 174 participants, 71 (40.8%) were males, median age was 32 years for the HbSS and HbAA groups and 46 years for the HbAS group (Table S1).

RBC deformability was measured by the laser optical rotational red cell analyzer (Lorrca, RR Mechatronics) [5] as the elongation index (EI) under three assay conditions: Increasing shear stress (deformability assay), continuous changes in osmotic gradient (osmoscan), and decreasing oxygen tension (oxygenscan assay) [6, 7]. The point of sickling (PoS), the oxygen partial pressure at which RBCs begin to sickle, was derived from the oxygenscan assay.

We tested associations between EI and hemolytic markers (lactate dehydrogenase [LDH], total bilirubin) in the HbSS group (biochemical data were unavailable for HbAS/HbAA). We also examined independent and combined effects of HbF level, coinherited α‐globin genotype, HU therapy, and the PIEZO1 E756del variant on EI across genotypes. Alpha‐globin genotyping was available for 58/72 (80%) HbSS, 57/68 (84%) HbAS, and 32/34 (94%) HbAA participants (Table S2). Two HbAA participants with ⍺⍺/⍺⍺⍺ genotype were excluded due to the small number of subjects in that group and HbAS participants with −⍺/−⍺ or −⍺/⍺⍺ were combined into a single α‐thalassemia group. PIEZO1 E756Del genotyping was available for 69 HbSS, 58 HbAS, and 23 HbAA participants. Statistical analysis included Dunn's test for pairwise group comparisons, Wilcoxon rank‐sum test for two‐group comparisons, Spearman's correlation for continuous associations, and linear regression to assess the effects of relevant covariates.

3. Results

HbSS RBCs show markedly reduced deformability across all conditions. HbSS RBCs showed lower deformability (lower EI) at all shear stress levels and across osmotic and oxygen gradients, and demonstrated greater inter‐individual variability, compared to HbAS and HbAA (Figure 1A–C). Pairwise comparison showed significant differences between HbSS and both HbAS and HbAA across essentially all shear stress levels (p < 0.00001), with the sole exception of 0.3 Pa for HbSS versus HbAS (Table S3A). HbAS also differed significantly from HbAA at most, but not all, shear stress levels and osmoscan parameters (Table S3A).

FIGURE 1.

Red cell deformability among individuals with different HBB genotypes. (A) Deformability of red cells from each individual in the HbSS, HbAS, and HbAA groups. (B) Summary (mean with standard deviation) of deformability for HbSS, HbAS, and HbAA groups. (C) Individual graphs per genotype in the osmoscan assay.

Reduced deformability correlates with hemolysis in HbSS. Across all shear stress levels, lower EI (reduced deformability) was significantly correlated with increased LDH and total bilirubin, indicating greater hemolysis (Figure 2; Table S3B). PoS, defined as the partial pressure of oxygen at which RBCs start sickling, was positively correlated with both LDH (ρ = 0.41, p‐value = 0.0027) and total bilirubin (ρ = 0.47, p‐value = 0.0005) (Table S3B), indicating that RBCs prone to earlier sickling were associated with greater hemolysis.

FIGURE 2.

Negative correlation of elongation index (EI) with LDH at 3 Pa (A) and 30 Pa (B); with total bilirubin at 3 (C) and 30 Pa (D).

HbF, but not α‐thalassemia, independently improves deformability. HbF level correlated positively with EI at 3 Pa (ρ = 0.494, p‐value < 0.0002; Figure S1A), and negatively with PoS (ρ = −0.434, p‐value = 0.001; Figure S1B), confirming that higher HbF both improves deformability and delays sickling onset. Dividing the HbSS cohort into low (HbF ≤ 5%) and high (HbF > 20%) groups showed significantly better deformability across all shear stress levels in the high‐HbF group (p < 0.01; Figure S2, Table S4).

In contrast, compared to wild type (⍺⍺/⍺⍺), subjects with coinherited α‐thalassemia showed no significant association with deformability for HbAS and HbSS, or HbAA (except at EI min (p = 0.039) and O hyper (p = 0.0001) (Table S5A–C; Figure S3A–C). In HbSS, α‐thalassemia was associated with higher median hematocrit (p = 0.091), hemoglobin (p = 0.113), and RBC count (p = 0.000), but lower MCHC (p = 0.040), MCV (p = 0.002), absolute reticulocytes (p = 0.451), total bilirubin (p = 0.321), direct bilirubin (p = 0.225), and LDH (p = 0.436), but none of these hematological shifts translated into a significant deformability difference (Table S5A). Combined analysis of HbF and α‐thalassemia status confirmed that HbF level, not α‐thalassemia, was the consistent independent predictor of improved deformability (Table S6; Figure S4).

HU improves deformability. Participants on HU therapy showed significantly higher EI across all shear stresses, higher EI max (p = 0.0002) and EI hyper (p = 0.0002) on osmoscan. On oxygenscan, those on HU also had significantly higher EI min (p = 0.010) and EI max (p = 0.0003) with a lower PoS (p = 0.009) (Table S7 ; Figure S5), indicating both improved deformability and delayed sickling onset with HU treatment.

PIEZO1 E756del reduces deformability in HbSS. We assessed whether PIEZO1 E756Del affected Lorrca parameters in 69 HbSS, 58 HbAS, and 23 HbAA participants. Among HbSS participants, those carrying PIEZO1 E756Del gain‐of‐function variant showed significantly reduced EI under increasing shear stress, osmotic pressure, and decreasing oxygen pressure, along with lower O‐min (measure of osmotic fragility), and a rightward shift in O‐hyper (hypotonic osmolality) (Figure S6A–C). In HbAS and HbAA individuals, PIEZO1 E756Del did not affect EI under shear stress but was associated with lower O‐min, consistent with PIEZO1 E756Del driving the RBC dehydration and resistance to hypotonic lysis (Figure S7A–D).

4. Discussion

This study demonstrates clear, genotype‐dependent differences in RBC deformability across HbSS, HbAS, and HbAA, most pronounced under increased shear stress and osmotic stress (Figure 1A–C). Within HbSS, reduced deformability tracked closely with hemolytic markers, reinforcing ektacytometry's utility as a functional readout of RBC health in SCD (Figure 2).

Notably, our data show that elevated HbF — independent of α‐thalassemia status—was the dominant modifier of improved deformability, consistent with the well‐established clinical observation that SCD patients maintaining HbF above ∼20% experience milder disease, fewer hospitalizations, and improved survival [8, 9]. In contrast, coinherited α‐thalassemia did not independently improve deformability in our cohort, despite producing the expected hematological changes (higher RBC count and hematocrit, lower MCV/MCHC). This finding contrasts with some prior reports linking α‐thalassemia to improved sickle RBC rheology [10–13], and may reflect differences in cohort composition, HU treatment prevalence, or the specific deformability parameters measured; it underscores that the relationship between α‐thalassemia and RBC rheology in SCD is more complex, and less independent of other genetic and therapeutic modifiers, than commonly assumed. HU therapy produced a clear, consistent improvement in deformability across all assay types, consistent with prior work [12]. This is likely mediated substantially through HU's HbF‐inductive effect, though direct membrane or hydration effects cannot be excluded from this analysis.

PIEZO1 is a mechanosensitive cation channel that modulates intracellular calcium (Ca²⁺) influx and RBC volume [14]. Ex vivo, direct pharmacological activation of PIEZO1 significantly decreases SCD RBC deformability [15]. However, PIEZO1‐mediated Ca²⁺ entry under mechanical stress requires extracellular Ca²⁺ [16], and standard ektacytometry buffers are nominally Ca²⁺‐free; the shear stress applied in our assay therefore cannot itself trigger acute PIEZO1‐dependent Ca²⁺ influx. We interpret the reduced deformability in PIEZO1 E756del in HbSS carriers as most likely reflecting a pre‐existing elevation in basal intracellular Ca²⁺, accrued in vivo prior to sample collection, rather than a real‐time channel effect occurring during the measurement itself [15, 17]. Given the high prevalence of this variant in populations of African descent [18], it may still represent an underappreciated contributor to the substantial inter‐individual variability in RBC deformability and disease severity in SCD [17]. The lower O min in HbAS and HbAA E756del carriers is consistent with reports of osmotic fragility changes in E756del carriers, though evidence for a dehydration phenotype in heterozygotes has been mixed across studies, potentially reflecting differences in assay methodology or genetic background.

Limitations of our study include the modest sample size, particularly for genotype subgroup analyses (e.g., α‐thalassemia and PIEZO1 strata), and the unavailability of hemolytic marker data for HbAS and HbAA groups, precluding cross‐genotype correlation analyses.

In conclusion, this study confirms Lorrca ektacytometry as a robust and reliable tool for assessing RBC health in SCD [19], and clarifies that HbF level and HU therapy, rather than α‐thalassemia, are the dominant modifiers of RBC deformability in this cohort. The association between PIEZO1 E756del and reduced deformability in HbSS suggests that this common variant merits further study as a genetic modifier of SCD severity [17], and may offer additional insight into individual in treatment response to therapies such as pyruvate kinase activators.

Author Contributions

Xunde Wang was responsible for deformability studies, data collection, and creating the manuscript. Nancy Asomaning was responsible for data collection, preparation, analysis and editing the manuscript. Swee lay Thein supervised the project and edited the manuscript. Haiou Li performed statistical analyses. Maureen Lundt performed deformability studies and collected data. The final version of the manuscript was approved by all authors for publication.

Funding

This work was supported by the Division of Intramural Research of the National Heart, Lung, and Blood Institute. NIH, Department of Health and Human Services (DHHS). The funder had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. The opinions expressed in this article are those of the authors and do not necessarily reflect the views of the Department of Health and Human Services or NIH.

Ethics Statement

The protocol (NCT03685721) was approved by the NHLBI Institutional Review Board, and all participants provided written informed consent.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: jha270416‐sup‐0001‐SuppMat.pdf.

Data Availability Statement

Deidentified individual participant data in the reported results will be made available 3 months after publication for a period of 5 years after the publication date. Original data will be available from the corresponding author, Swee Lay Thein (sweelay.thein@nih.gov). Original data are available at dbgap.ncbi.nlm.nih.gov/home.