Sickle Cell Disease is the most common monogenic disease worldwide and represents a major public health concern in sub-Saharan Africa, where nearly 240,000 affected children are born each year [1][2]. Despite significant advances achieved in high-income countries, mortality and morbidity associated with this disease remain particularly high across the African continent due to delayed diagnosis, limited access to specialized healthcare, and the lack of adequate biological monitoring tools for disease progression [1].

The pathophysiology of sickle cell disease is now recognized as complex and multifactorial. Beyond hemoglobin S polymerization, several biological mechanisms contribute to the onset of vaso-occlusive crises, including oxidative stress, chronic inflammation, and tissue hypoxia [3][4]. These mechanisms contribute to progressive endothelial dysfunction responsible for the acute and chronic complications observed in sickle cell patients.

Oxidative stress plays a central role in this pathophysiology. Chronic hemolysis observed in sickle cell disease promotes excessive production of reactive oxygen species, leading to impairment of cellular antioxidant defense systems [5]. Several recent African and international studies have demonstrated increased levels of lipid peroxidation markers such as malondialdehyde (MDA), associated with reduced antioxidant defenses in HbSS patients, particularly during vaso-occlusive crises [5][6].

In parallel, chronic systemic inflammation plays a major role in amplifying vaso-occlusive processes. Increased levels of pro-inflammatory cytokines, particularly interleukin-6 (IL-6), as well as C-reactive protein (CRP), contribute to worsening vascular injury and ischemia-reperfusion phenomena [7]. Several recent studies have also reported markedly elevated levels of these inflammatory biomarkers during painful sickle cell episodes [8].

Over recent years, increasing interest has been directed toward molecular biomarkers involved in the regulation of oxidative stress. Among these, Forkhead box O3 (FoxO3) transcription factor appears to be an essential regulator of cellular antioxidant response and redox homeostasis [9]. FoxO3 is involved in controlling cellular defense mechanisms against oxidative stress and may represent a potential biomarker of severity in sickle cell disease. However, African data regarding its involvement in vaso-occlusive crises remain limited.

In sub-Saharan Africa, biological tools capable of improving biological characterization and diagnosis of vaso-occlusive crises remain insufficiently developed [2]. The identification of a biological profile combining oxidative, inflammatory, and metabolic biomarkers could improve clinical risk stratification and optimize the management of sickle cell patients [8][10]. In this context, the development of diagnostic models based on accessible biomarkers represents an innovative and particularly relevant approach in resource-limited settings.

The present study therefore aimed to develop a diagnostic model for vaso-occlusive crises in sickle cell patients based on biomarkers involved in oxidative stress and inflammation, including FoxO3, IL-6, CRP, lactate, and malondialdehyde (MDA). This work could contribute to a better understanding of the pathophysiological mechanisms of sickle cell disease in the African context and open perspectives for biomarker-based diagnostic approaches adapted to local realities.

The aim of our study was to develop a biological model capable of discriminating vaso-occlusive crises based on oxidative, inflammatory, and metabolic biomarkers in Congolese sickle cell subjects. The main findings showed that HbSS patients experiencing vaso-occlusive crises had significantly higher concentrations of FoxO3, IL-6, CRP, lactate, and MDA compared with HbAA, HbAS, and HbSS patients in steady state. The multivariate model developed from FoxO3, IL-6, CRP, lactate, and MDA demonstrated excellent diagnostic performance, with an area under the ROC curve of 0.91.

In our study, HbSS patients in vaso-occlusive crisis exhibited a higher annual frequency of crises and significantly higher pain scores than HbSS patients in steady state. These findings confirm the severe nature of vaso-occlusive episodes in sickle cell disease. Several African studies have shown that painful crises remain the leading cause of hospitalization and morbidity among sickle cell patients [11][12]. The pathophysiology of these episodes involves complex mechanisms combining hemoglobin S polymerization, tissue hypoxia, chronic inflammation, and oxidative stress [13].

The absence of significant differences in age and sex distribution across the four groups strengthens the comparability of our study populations and supports the validity of subsequent biomarker comparisons.

Our results demonstrated a progressive increase in serum FoxO3 concentrations from HbAA subjects to HbSS patients in vaso-occlusive crisis. Mean concentrations increased from 6.2 ± 1.4 ng/mL in HbAA subjects to 16.8 ± 3.5 ng/mL in HbSS patients during crisis. Although FoxO3 is mainly an intracellular transcription factor, several studies suggest that extracellular release may occur during intense cellular stress and systemic inflammatory processes. The increase observed in our study may reflect a compensatory activation of cellular pathways involved in oxidative stress regulation. FoxO3 plays an important role in redox homeostasis and in the regulation of cellular antioxidant mechanisms [7]. Currently, few African data are available regarding this biomarker in sickle cell disease, which gives particular importance to our study.

The linear regression analysis provided additional quantitative insights. FoxO3 alone explained 66% of MDA variance (R² = 0.66, β = 0.48, p < 0.001) and 55% of lactate variance (R² = 0.55, β = 0.20, p < 0.001). These findings indicate a strong dose-dependent relationship between FoxO3 and oxidative stress markers. The striking difference between the R² for MDA (66%) and that for CRP (25%) suggests that FoxO3 is more tightly coupled to lipid peroxidation pathways than to systemic inflammatory response in sickle cell disease. MDA, as the end product of polyunsaturated fatty acid peroxidation, may directly reflect the oxidative burden resulting from chronic hemolysis and ischemia-reperfusion injury, whereas CRP is influenced by a broader range of inflammatory stimuli beyond oxidative stress.

IL-6 and CRP concentrations were significantly higher in HbSS patients during vaso-occlusive crises. Median IL-6 concentrations reached 18.7 [14.5 - 26.9] pg/mL in patients during crisis compared with 8.6 [6.2 - 11.4] pg/mL in HbSS patients in steady state. Similarly, median CRP concentrations were markedly increased in patients during crisis. These findings confirm the presence of a significant systemic inflammatory state during vaso-occlusive crises. Studies conducted in Ghana and other African countries have also reported significantly increased pro-inflammatory cytokines and inflammatory markers in symptomatic sickle cell patients [14][15]. Chronic inflammation contributes to cellular adhesion, ischemia-reperfusion injury, and worsening of vaso-occlusive phenomena [16].

The linear regression analysis revealed that FoxO3 explained 32% of IL-6 variance (R² = 0.32, β = 1.45, p < 0.001) and 25% of CRP variance (R² = 0.25, β = 1.80, p < 0.001). Although these associations were statistically significant, they were less robust than those observed for MDA and lactate, suggesting that FoxO3 is more directly involved in oxidative stress pathways than in the inflammatory cascade. This finding is consistent with the primary role of FoxO3 as a regulator of cellular antioxidant defense rather than a direct mediator of inflammation.

Malondialdehyde (MDA), the main marker of lipid peroxidation, was markedly increased in HbSS patients during vaso-occlusive crises, with a mean concentration of 9.8 ± 2.1 µmol/L compared with 5.7 ± 1.4 µmol/L in HbSS patients in steady state. These findings are consistent with previous studies reporting increased oxidative stress, elevated MDA concentrations, and impaired antioxidant defenses in patients with sickle cell disease, particularly during vaso-occlusive crises [17][18]. The strong increase in MDA observed in our study further supports the central role of lipid peroxidation in the pathophysiology of vaso-occlusive events.

The exceptionally strong linear relationship between FoxO3 and MDA (R² = 0.66, ρ = 0.81) observed in our study reinforces the central role of oxidative stress in the pathophysiology of vaso-occlusive crises. This strong correlation suggests that FoxO3 and MDA are biologically interconnected, possibly through the activation of redox-sensitive signaling pathways. The dose-dependent nature of this relationship (β = 0.48) indicates that a 1 ng/mL increase in FoxO3 is associated with a 0.48 µmol/L increase in MDA, providing a quantitative basis for future studies aiming to establish diagnostic thresholds.

Lactate was also significantly increased in HbSS patients during vaso-occlusive crises. Mean lactate concentrations reached 5.1 ± 1.2 mmol/L in patients during crisis compared with 2.8 ± 0.7 mmol/L in patients in steady state. This elevation may reflect tissue hypoxia and anaerobic metabolism secondary to microvascular vaso-occlusive episodes. Our findings therefore suggest that lactate could represent an interesting metabolic biomarker for evaluating the clinical severity of vaso-occlusive crises. Several African authors have emphasized the importance of developing simple and accessible biomarkers adapted to resource-limited settings [19].

The linear regression analysis showed that FoxO3 explained 55% of lactate variance (R² = 0.55, β = 0.20, p < 0.001), ranking second only to MDA in terms of association strength. This finding suggests that oxidative stress and metabolic disturbances are closely intertwined in sickle cell disease. The strong correlation between FoxO3 and lactate (ρ = 0.74) may reflect the fact that tissue hypoxia, a major driver of lactate production, also triggers oxidative stress through ischemia-reperfusion mechanisms.

Multivariate analysis identified FoxO3, IL-6, CRP, lactate, and MDA as independent factors significantly associated with vaso-occlusive crises. Lactate (OR = 2.11) and MDA (OR = 1.74) showed the strongest associations, followed by FoxO3 (OR = 1.48), IL-6 (OR = 1.27), and CRP (OR = 1.18). These findings reinforce the hypothesis that oxidative stress, systemic inflammation, and metabolic disturbances play complementary roles in the pathophysiology of vaso-occlusive crises. To our knowledge, few studies worldwide, and even fewer from Africa, have simultaneously integrated oxidative, inflammatory, and metabolic biomarkers into a diagnostic model for vaso-occlusive crises. Our findings therefore provide additional evidence supporting the use of multimarker approaches for biological risk stratification in sickle cell disease. Although moderate collinearity was observed between CRP and IL-6 (ρ = 0.61), both biomarkers were retained in the final model because of their independent clinical relevance and statistical significance. The inclusion of CRP provided complementary information on the systemic inflammatory response, whereas IL-6 reflected a more specific pro-inflammatory signaling pathway. Variance inflation factors for all five biomarkers ranged from 2.9 to 4.1, confirming acceptable levels of collinearity.

The biological model developed in this study demonstrated excellent diagnostic performance, with an area under the ROC curve of 0.91 (95% CI: 0.85 - 0.96), sensitivity of 88.9% (95% CI: 76.0 - 96.3), and specificity of 84.4% (95% CI: 70.5 - 93.5). These performances suggest that a multiparametric approach combining FoxO3, IL-6, CRP, lactate, and MDA could improve the early identification of patients at risk of vaso-occlusive crises. Recent studies have also highlighted the growing interest in circulating biomarkers in precision medicine approaches applied to sickle cell disease in Africa [20].

In our study, hydroxyurea use was more frequent among HbSS patients in steady state (64.4%) than among those experiencing vaso-occlusive crises (40.0%). This observation may reflect the protective effect of hydroxyurea in preventing painful episodes. Several international studies have confirmed the effectiveness of hydroxyurea in reducing the frequency of vaso-occlusive crises [10][21]. The lower proportion of hydroxyurea users in the crisis group may also indicate poor therapeutic adherence or inadequate dosing, which are common challenges in resource-limited settings.

To address the potential confounding effect of hydroxyurea, we performed two sensitivity analyses. First, we included hydroxyurea use as a covariate in the multivariate logistic regression model; all five biomarkers remained significantly associated with vaso-occlusive crises after this adjustment (Table 3). Second, we conducted a subgroup analysis restricted to the 43 HbSS patients not receiving hydroxyurea, in which the associations between biomarkers and crisis status remained significant and of similar magnitude (FoxO3: OR = 1.52, p = 0.003; IL-6: OR = 1.31, p = 0.007; CRP: OR = 1.21, p = 0.015; lactate: OR = 2.24, p = 0.002; MDA: OR = 1.82, p = 0.003). These findings indicate that the observed biomarker differences are not merely attributable to hydroxyurea treatment but reflect intrinsic pathophysiological differences between steady-state and crisis patients. Nevertheless, the protective effect of hydroxyurea was confirmed in our adjusted model (adjusted OR = 0.42, p = 0.006), consistent with its established role in reducing VOC frequency.

Nevertheless, this study has certain limitations. Its cross-sectional design does not allow the establishment of a causal relationship between the studied biomarkers and the occurrence of vaso-occlusive crises, nor does it permit prediction of future crises. The sample size remains relatively modest and monocentric, limiting the generalizability of the findings. The absence of longitudinal follow-up does not allow assessment of the dynamic variations of biomarkers over time or confirmation of the true long-term prognostic value of the model. Furthermore, due to the lack of an external validation cohort or internal validation procedure (e.g., bootstrap resampling or k-fold cross-validation), the reported AUC, sensitivity, and specificity represent apparent performance estimates obtained on the development sample. These estimates may be optimistic and should be interpreted cautiously.

Additionally, FoxO3 is primarily an intracellular transcription factor, and its measurement in serum requires further validation as a reliable circulating biomarker. The extracellular release mechanisms of FoxO3 during cellular stress remain incompletely understood. Future studies should explore correlations between serum FoxO3 levels and its intracellular expression in relevant cell types (e.g., erythrocytes, endothelial cells, or leukocytes).

Despite these limitations, this work represents one of the first Congolese studies simultaneously integrating FoxO3, IL-6, CRP, lactate, and MDA into a biological model associated with vaso-occlusive crises in African sickle cell subjects. The excellent diagnostic performance of the model (AUC = 0.91) supports its potential utility in clinical settings, particularly in resource-limited environments where access to specialized hematological care is restricted.

Recent studies have highlighted the increasing value of multiparametric models integrating clinical and biological biomarkers for improving the identification and risk stratification of vaso-occlusive crises in patients with sickle cell disease [22]-[25]. In agreement with these reports, our model demonstrated excellent apparent diagnostic performance (AUC = 0.91), supporting the potential usefulness of combining oxidative, inflammatory, and metabolic biomarkers for biological classification of vaso-occlusive crises. Nevertheless, external validation in independent cohorts remains necessary before clinical implementation.

Longitudinal multicenter studies including larger sample sizes will be necessary to validate these findings, evaluate the stability of the model over time, and clarify the clinical relevance of these biomarkers in the follow-up of African sickle cell patients. Prospective studies could also assess whether baseline levels of FoxO3, MDA, or lactate predict the future occurrence of vaso-occlusive crises, which would strengthen the prognostic value of the model.

Furthermore, the strong linear relationship between FoxO3 and MDA (R² = 0.66) raises the possibility that FoxO3 could serve as a surrogate biomarker for oxidative stress severity when direct MDA measurement is unavailable. This is particularly relevant in resource-limited settings where advanced laboratory equipment may be lacking. Simple point-of-care assays targeting FoxO3 or its downstream effectors could be developed for routine clinical use.

This study developed a biological model discriminating vaso-occlusive crises based on oxidative, inflammatory, and metabolic biomarkers in Congolese sickle cell subjects. HbSS patients during crisis had significantly higher concentrations of FoxO3, IL-6, CRP, lactate, and MDA compared with HbAA, HbAS, and HbSS steady-state patients.

Linear regression analysis revealed a strong dose-dependent relationship between FoxO3 and oxidative stress markers. FoxO3 alone explained 66% of MDA variance (R² = 0.66, β = 0.48, p < 0.001) and 55% of lactate variance (R² = 0.55, β = 0.20, p < 0.001), but only 25% of CRP variance, suggesting that FoxO3 is more directly involved in oxidative stress than in systemic inflammation.

Multivariate logistic regression identified FoxO3, IL-6, CRP, lactate, and MDA as independent factors associated with vaso-occlusive crises. Lactate (OR = 2.11) and MDA (OR = 1.74) showed the strongest associations, followed by FoxO3 (OR = 1.48), IL-6 (OR = 1.27), and CRP (OR = 1.18). The combined model demonstrated excellent diagnostic performance (AUC = 0.91, 95% CI: 0.85 - 0.96; sensitivity = 88.9%, 95% CI: 76.0 - 96.3; specificity = 84.4%, 95% CI: 70.5 - 93.5), confirming the major role of oxidative stress, inflammation, and metabolic disturbances in sickle cell crises. The strong linear relationship between FoxO3 and MDA (R² = 0.66) suggests that FoxO3 could serve as a surrogate biomarker for oxidative stress severity when direct MDA measurement is unavailable, particularly in resource-limited African settings.

Sensitivity analyses confirmed that the associations between biomarkers and vaso-occlusive crises persisted after adjustment for hydroxyurea use and comorbidities, as well as in the subgroup of patients not receiving hydroxyurea, indicating that the observed biomarker differences reflect intrinsic pathophysiological differences rather than treatment effects.

Despite limitations related to the cross-sectional and monocentric design, and the lack of external validation, this work represents one of the first Congolese studies integrating these biomarkers into a diagnostic model for vaso-occlusive crises. The cross-sectional nature of this study precludes establishing causality or predicting future crises; rather, the model provides a biological signature for discriminating between steady-state patients and those experiencing an ongoing crisis. Longitudinal multicenter studies are needed to validate these findings and assess the long-term prognostic value of the model.