Prevalence and predictors of splenic dysfunction in Yemeni children with sickle cell anemia: a cross-sectional study

¹Hematology and Internal Medicine Department, Faculty of Medicine, Taiz University of Medical Sciences, Taiz, Yemen, ²Department of Internal Medicine, Saeed Thabet Nasher Hospital, Taiz, Yemen, ³Urology Research Center, Al-Thora General Hospital, Department of Urology, School of Medicine, Ibb University of Medical Sciences, Ibb, Yemen, ⁴Registrar in Hematology and Internal Medicine Department, Dr. Saeed Thabet Nasher Hospital, Taiz, Yemen, ⁵Internal Medicine Department, Faculty of Medicine, Taiz University of Medical Sciences, Taiz, Yemen

^&Corresponding author
Faisal Ahmed, Registrar in Hematology and Internal Medicine Department, Dr. Saeed Thabet Nasher Hospital, Taiz, Yemen

Introduction    

Sickle cell anemia (SCA) is a devastating monogenic hemoglobinopathy defined by the production of abnormal hemoglobin (HbS). This molecular defect causes erythrocytes to undergo polymerization and assume a rigid, sickle-like configuration under hypoxic conditions [1]. The resulting cascade of chronic hemolysis and microvascular occlusion drives a progressive multisystem disorder [1]. Among the earliest and most profound complications is the loss of splenic integrity, often referred to as autosplenectomy [2]. The specialized microvasculature of the splenic red pulp facilitates the entrapment of sickled cells, leading to recurrent micro-infarctions, parenchymal scarring, and eventually, the complete loss of the organ's filtration capacity [2]. This functional asplenia creates a critical immune deficit, leaving patients, particularly children, highly susceptible to life-threatening sepsis caused by encapsulated pathogens such as Streptococcus pneumoniae [3].

In high-income nations, the clinical narrative of splenic failure has been significantly improved through integrated public health strategies. The implementation of universal newborn screening, early penicillin prophylaxis, and the widespread use of hydroxyurea has successfully delayed splenic attrition and reduced associated mortality [4]. In contrast, children in resource-limited and conflict-affected regions like Yemen face persistent barriers to these essential standards of care [3]. Consequently, there is an urgent need for diagnostic tools that are both affordable and reliable to identify those at the highest risk for immune failure. While technetium-99m scintigraphy remains the definitive diagnostic standard, its reliance on nuclear medicine infrastructure and high costs render it inaccessible in most Yemeni clinical settings [5]. The detection of Howell-Jolly bodies (HJB) provides a pragmatic alternative. These nuclear remnants, which are typically cleared by a functional spleen, serve as a specific surrogate for advanced splenic dysfunction [1,6]. High HJB prevalence in pediatric SCA cohorts has demonstrated significant clinical utility for detecting hyposplenism in underserved populations [6,7].

The global literature highlights a striking geographic heterogeneity in the tempo of splenic decline. While children in Western cohorts often experience universal asplenia by age five [8], those in the Middle East and India frequently exhibit a more protracted course [9]. This relative preservation of splenic function is largely attributed to the protective influence of higher fetal hemoglobin (HbF) levels associated with the Arab-Indian ß-globin haplotype [10]. Despite the high burden of SCA in Yemen [11,12], there is currently a total absence of peer-reviewed data regarding the prevalence or clinical determinants of splenic dysfunction in this specific population. This study was therefore conducted to bridge this critical information gap and to identify modifiable risk factors that can guide local clinical management.

In this report, we evaluate the prevalence of splenic dysfunction through HJB screening among Yemeni children and determine the clinical and laboratory predictors associated with the loss of splenic function.

Methods     

Study design and setting

This retrospective cross-sectional study was conducted from June 2022 to December 2023 at Saeed Thabet Nasher Hospital-the primary tertiary referral center for hematological disorders serving children across Taiz, Yemen.

Participants

We consecutively enrolled children aged 1-10 years with confirmed homozygous sickle cell anemia (HbSS), diagnosed through high-performance liquid chromatography (HPLC; defined as HbS >90% with absent HbA). To ensure a clean assessment of red blood cell morphology, we excluded children with a prior splenectomy, co-inherited hemoglobinopathies (such as β-thalassemia), or a blood transfusion within the three months prior to enrollment (Figure 1).

Sample size and sampling

Given the absence of prior Yemen-specific data on splenic dysfunction in pediatric SCA, we performed an a priori sample size calculation to reliably estimate the prevalence of Howell-Jolly bodies (HJB), our primary surrogate marker. Drawing from Taiz's high βS gene frequency (>4%) [13] and reports from neighboring countries showing hyposplenia rates of 38-45% in pediatric SCA cohorts with similar genetic backgrounds [14-16], we anticipated a 40% prevalence (P= 0.40). Using the single population proportion formula:

Where: n: required sample size; Z: Z-statistic for 95% confidence level (Z = 1.96); p: expected prevalence of splenic dysfunction (0.40, based on regional data of 38-45%); d: margin of error (0.15). Substituting values:

Over the 18-month study period, consecutive enrollment at this single center yielded a final cohort of 41 eligible children, meeting our calculated target. The observed prevalence of 18 HJB-positive cases (43.9%) provided an event per variable (EPV) ratio >10 for up to two key predictors in subsequent exploratory regression analyses, adhering to recommended thresholds for hypothesis-generating research [17].

Ethical considerations

The study received ethical approval from the Institutional Review Board of Taiz University of Medical Sciences (Ref: TUMS/IRB/2022-15). Written informed consent was obtained from the parent or legal guardian of each participant. All procedures respected participant confidentiality.

Variables and data sources

Primary outcome: splenic dysfunction, operationally defined as the presence of one or more Howell-Jolly bodies (HJB) per 1000 red blood cells on a peripheral blood smear, a validated surrogate for impaired splenic phagocytic function [18,19].

Exposures and covariates: data collected included demographic factors (age, sex), clinical history (frequency of vaso-occlusive crises, SCA-related hospitalizations, history of blood transfusions, frequent infections defined as =3 physician-diagnosed episodes requiring antibiotics in the prior year, and use of disease-modifying therapies like hydroxyurea), physical examination findings (splenomegaly, defined as a spleen palpable =1 cm below the left costal margin), and laboratory parameters (complete blood count, reticulocyte count, and hemoglobin fractions via HPLC).

Data collection procedures

All participants underwent a standardized assessment. Detailed clinical histories were obtained from parents/guardians and supplemented with medical record review. A focused physical examination was performed. Laboratory analyses included a complete blood count and reticulocyte count (Sysmex XN-1000 analyzer) and hemoglobin fraction quantification (Bio-Rad Variant II HPLC). For the primary outcome, air-dried peripheral blood smears were stained with Wright-Giemsa and examined under oil immersion (1000x magnification) by two independent hematologists blinded to all clinical data. Each evaluator assessed a minimum of 1000 erythrocytes. HJBs were identified as characteristic small, round, dense, purple intracytoplasmic inclusions. A smear was considered positive if at least one definitive HJB was identified. Inter-observer agreement prior to consensus was excellent (Cohen's kappa = 0.85). Discrepant readings were resolved through joint review. Abdominal ultrasonography was performed to document spleen size and detect structural abnormalities; however, sonographic metrics were not used to define the primary outcome.

Statistical analysis

Analyses were performed using SPSS Statistics v21.0 (IBM Corp.). Continuous data were assessed for normality using the Shapiro-Wilk test. Normally distributed variables are presented as mean ± standard deviation and compared using the independent Student's t-test. Non-normally distributed variables are summarized as median (interquartile range) and compared using the Mann-Whitney U test. Categorical variables are presented as frequency (%) and analyzed using Fisher's exact test.

Variables associated with HJB positivity at a significance level of p < 0.10 in univariate analyses were entered into a multivariate binary logistic regression model using backward stepwise selection (retention criterion: p = 0.05). Results are reported as adjusted odds ratios (aOR) with 95% confidence intervals (CI). Given the exploratory nature of this study and the need to avoid premature exclusion of potential predictors, we employed a liberal entry threshold (p<0.10), acknowledging this increases the risk of Type I error. Model performance was evaluated by calculating the area under the receiver operating characteristic curve (AUC) for discrimination and the Hosmer-Lemeshow test for calibration. Multicollinearity was assessed using variance inflation factors (all <2). Statistical significance for final model inferences was set at a two-tailed p < 0.05.

Results     

Cohort characteristics

A total of 41 children with homozygous SCA (HbSS) were enrolled, with a mean age of 4.95 years (SD 2.3). The cohort was characterized by significant disease morbidity: 46.3% (n=19) of the children suffered from frequent infections and 31.7% (n=13) reported recurrent vaso-occlusive crises. Notably, despite this high clinical burden, hydroxyurea utilization was limited to 12.2% (n = 5) of the participants.

Prevalence of splenic dysfunction

Splenic dysfunction, identified by the presence of Howell-Jolly bodies (HJB) on peripheral blood smears, was present in 18 children, representing a prevalence of 43.9% (95% CI 28.5-59.3%).

Comparative analysis: HJB-positive vs. HJB-negative groups

Statistical comparison between HJB-positive and HJB-negative groups revealed several significant differences in clinical and laboratory profiles (Table 1). Children with splenic dysfunction were significantly older (mean 6.1 vs. 4.1 years, p=0.005) and more likely to be over the age of five (p = 0.016). From a clinical burden perspective, the HJB-positive group showed a higher frequency of intensive medical interventions, including more than five lifetime blood transfusions (50.0% vs. 13.0%, p=0.009) and a history of more than three hospitalizations (50.0% vs. 21.7%, p=0.059). Laboratory analysis showed that an HbS fraction exceeding 70% was significantly more common in the dysfunction group (66.7% vs. 30.4%, p=0.020). Conversely, the presence of a palpable spleen did not differ significantly between the two groups (p = 0.715).

Predictors of splenic dysfunction

Multivariate logistic regression identified five independent predictors of HJB-positive status (Table 2). The most potent clinical predictor was the history of more than five transfusions (aOR 5.2, 95% CI 1.4-19.1, p = 0.015). Significantly, the absence of hydroxyurea therapy emerged as a critical modifiable predictor (aOR 4.9, 95% CI 1.4-17.2, p = 0.013). Other independent predictors included an HbS fraction greater than 70% (aOR 4.5, p = 0.019), age over five years (aOR 4.1, p = 0.024), and more than three prior hospitalizations (aOR 3.8, p = 0.038). The model demonstrated robust discriminatory ability with an Area Under the Curve (AUC) of 0.87 (95% CI 0.76 - 0.98).

Ethics approval and consent to participate

This retrospective cohort study was approved by the Institutional Review Board of University (Reference: IRB-IU-2025-12). The study was conducted in accordance with the Declaration of Helsinki. Due to the retrospective nature and anonymization of data, individual patient consent was waived.

Discussion     

Principal findings

This study provides a systematic evaluation of splenic status in Yemeni children with SCA. Our primary finding is that nearly half (43.9%) of children aged 1-10 years exhibit functional hyposplenia. We identified a high-risk phenotype characterized by advancing age, higher hospital burden, and an HbS fraction >70%. Most importantly, the lack of hydroxyurea therapy emerged as the strongest independent predictor of organ failure, reflecting a modifiable crisis in patient care.

Evaluating functional splenic dysfunction in sickle cell anemia

Assessing functional splenic dysfunction in SCA is crucial for risk stratification, yet it requires balancing diagnostic precision with practical feasibility in resource-limited settings. Technetium-99m scintigraphy using heat-damaged erythrocytes provides gold-standard sensitivity for splenic phagocytic capacity, but high costs, invasiveness, and nuclear facility needs limit access where SCA affects children most [20,21]. In this study we used Howell-Jolly bodies (HJBs) on peripheral blood smears for assessing the splenic function; this method offer a simple, low-cost alternative using standard microscopy; they are highly specific (96.7%) for confirming advanced dysfunction but less sensitive (35.0%) for early detection [18]. Pitted red cell counts via interference phase microscopy [22,23] and emerging flow cytometry assays for high-mannose glycans on erythrocytes (93% sensitivity, 100% specificity) [23,24] detect subtler impairment but demand specialized equipment. In resource-strapped health systems, HJB screening serves as a pragmatic mainstay, prioritizing accessibility to safeguard at-risk patients.

Prevalence and age-related patterns

The observed prevalence of HJB-positive functional hyposplenia (43.9%) is substantially lower than that reported in Western cohorts, where functional asplenia approaches 90-100% by age five [25,26]. This finding aligns with reports from other Middle Eastern and South Asian populations, where splenic dysfunction develops more gradually [18,26,27]. For example, similar prevalence rates have been reported in Oman (38%) and Saudi Arabia (45%) [14,28]. The delayed progression in these regions has been attributed to the influence of the Arab-Indian ß-globin haplotype and co-inherited alpha-thalassemia, both of which are associated with higher HbF levels and milder hemolytic profiles, potentially preserving splenic tissue for longer periods [10]. However, due to the single-center design and relatively small sample size in the present study, these findings should be interpreted with caution, and further multi-center studies with larger cohorts are warranted to validate these results and assess their broader applicability. Age-stratified analyses (e.g., <5 vs. 5-10 years) further support this protracted course, though exact rates require confirmation in prospective designs.

The milder and more protracted course of splenic dysfunction observed in Yemen mirrors findings from other regions with similar genetic backgrounds [29,30]. For example, in Indian cohorts, functional asplenia may be delayed until adolescence, attributed to the protective effects of higher HbF [31]. However, despite this relative delay, the clinical burden of SCA remains high, with substantial rates of infection (46.3%) and vaso-occlusive crises (VOC; 31.7%)-findings echoed in the current study. This suggests that even partial preservation of splenic function does not eliminate the risk of SCA complications, highlighting the need for comprehensive care regardless of phenotype.

Clinical and laboratory predictors

In this study, multivariable statistical modeling elucidated significant independent predictors of functional hyposplenia: older age, recurrent hospital admissions and blood transfusions, elevated HbS percentage, heightened lactate dehydrogenase levels, and the absence of hydroxyurea therapy. These determinants align with established markers of disease severity while highlighting the profound impact of therapeutic access, offering a nuanced understanding of disease progression in a resource-constrained setting [24,32].

Older age is a consistent predictor of splenic impairment, attributed to cumulative microinfarction [32]. While the BABY HUG trial in the United States demonstrated a rapid decline in splenic filtration function in early childhood [26], our Yemeni cohort exhibited a more protracted course. This may be attributable to the high prevalence of the Arab-Indian (Benin) ß-globin haplotype, which is associated with elevated fetal hemoglobin (HbF) levels and a milder hemolytic phenotype compared to the African haplotypes predominant in Western cohorts [10,33].

Recurrent hospitalizations and blood transfusions are robust indices of a severe disease phenotype, characterized by frequent vaso-occlusive crises and chronic anemia that accelerate end-organ damage [34]. These aligns with findings from other Middle Eastern populations, such as Oman and Saudi Arabia, where severe clinical courses are linked to earlier splenic dysfunction [14,28]. Elevated HbS percentage (>70% in our model) is a direct biochemical marker of severity, increasing intracellular HbS polymer formation and the propensity for sickling and ischemic injury [35]. This association is consistent across diverse populations, underscoring HbS level as a fundamental driver of pathophysiology [36].

Most critically, the underutilization of hydroxyurea emerged as a powerful, modifiable predictor. Only 12.2% of our cohort received this disease-modifying therapy, a stark contrast to uptake rates in clinical trial and high-income settings where its efficacy in raising HbF and reducing complications is proven [37,38]. This access disparity, against a backdrop of high infection rates (46.3%), represents a key target for intervention. Hydroxyurea has been shown to improve splenic function parameters and delay functional asplenia, making its scaled-up utilization a paramount public health priority for preserving organ function and reducing morbidity in Yemeni children [39,40].

Implications for clinical practice

The striking underutilization of hydroxyurea (12.2%) in this cohort underscores a critical therapeutic gap. In high-income settings, hydroxyurea is widely recommended for children with SCA, with robust evidence supporting its role in reducing VOC, transfusion needs, and preserving organ function [40,41]. Barriers to hydroxyurea use in Yemen likely include limited access, lack of provider and patient awareness, concerns about safety, and inadequate healthcare infrastructure [40,42]. Addressing these barriers could substantially alter the disease course for Yemeni children with SCA. Routine screening for HJB is feasible, low-cost, and can be integrated into standard care protocols even in resource-limited settings [19]. This approach could enable early identification of children at risk for splenic dysfunction, guiding targeted interventions such as intensified infection prophylaxis, prioritized vaccination, and prompt initiation of hydroxyurea therapy.

Study limitations

The primary limitation of this research is the cross-sectional design, which captures a single moment in time and cannot confirm the exact temporal sequence of splenic decline. Furthermore, HJB detection, while highly specific, is less sensitive than pitted red cell counts or scintigraphy; thus, our prevalence figures likely represent only the more advanced stages of hyposplenism. The modest sample size also means the odds ratios, while statistically significant, have wide confidence intervals. These factors suggest that while our findings are a strong signal for clinical practice, they should be applied cautiously until validated by larger national cohorts.

Conclusion     

Functional splenic failure affects a significant portion of Yemeni children with SCA, yet the progression is slower than Western norms, offering a critical window for intervention. Our findings identify a profound therapeutic gap: the underutilization of hydroxyurea is the most significant modifiable driver of early organ failure. To move forward, we advocate for the integration of HJB screening into national protocols to risk-stratify pediatric patients. Expanding access to hydroxyurea must become a public health priority to preserve splenic function and reduce the burden of infection-related mortality in Yemen.

Competing interests     

The authors declare no competing interests.

Authors' contributions     

All the authors have read and agreed to the final manuscript.

Tables and figure     

Table 1: baseline characteristics of yemeni children with sickle cell anemia, stratified by howell-jolly body status

Table 2: unadjusted and adjusted logistic regression analysis of predictors of splenic dysfunction (HJB-positivity)

Figure 1: participant enrollment flowchart: diagram illustrating the screening and enrollment process for children with sickle cell anemia (SCA) in the study. Of 52 children initially assessed, 11 were excluded based on predefined criteria. The remaining 41 children were enrolled and classified based on the presence of Howell-Jolly bodies (HJB) on peripheral blood smear

References