Neutrophil-to-Lymphocyte Ratio in Pediatric Sickle Cell Anemia: A Case-Control Study from Gezira State, Sudan

Study design and setting

A hospital-based case-control study was conducted at Wad Medani Pediatric Teaching Hospital in Gezira State, Sudan, covering the period from September 2022 to June 2024. The tertiary facility serves as a primary pediatric referral center for hematological disorders across Gezira State and neighboring agricultural regions.

Study population and eligibility criteria

The study enrolled 130 pediatric participants aged 1 to 18 years, comprising 80 confirmed SCA cases and 50 age- and sex-matched healthy controls. SCA diagnosis was verified through qualitative hemoglobin electrophoresis (HbSS genotype). Case participants were enrolled during acute clinical presentation or inpatient admission, capturing real-time hematological parameters during acute events. Healthy controls consisted of age-matched pediatric individuals without underlying hematological abnormalities, chronic inflammatory states, or active acute infections.

Inclusion criteria encompassed pediatric patients aged <18 years with documented HbSS disease whose parents or legal guardians provided written informed consent (with patient assent obtained when appropriate). Exclusion criteria comprised: (1) concomitant hemoglobinopathies or thalassemia traits; (2) co-existing chronic metabolic or systemic conditions (such as diabetes mellitus); (3) systemic antibiotic therapy initiated prior to blood sampling (to prevent confounding leukocyte profiles); and (4) hemolyzed, clotted, or insufficient blood specimens.

Sample size estimation

To ensure adequate statistical power for evaluating differences in mean NLR between pediatric SCA cases and healthy controls, a sample size calculation was conducted prior to study execution using two-sample mean comparison formulas (G*Power version 3.1.9.7). Based on preliminary pilot data and published literature on pediatric hemoglobinopathies [18,19], assuming an expected mean difference in NLR of 1.5 units between groups (SD ± 2.5), a two-sided significance level (α) of 0.05, and a statistical power (1 - β) of 80% (0.80), the minimum total sample size required was determined to be 112 participants (70 cases and 42 controls). To account for potential specimen loss or technical non-evaluability, the target sample size was expanded to 130 participants (80 confirmed SCA cases and 50 healthy controls). This final sample size provided a statistical power exceeding 85% to detect primary differences in NLR between cases and controls.

Laboratory analysis

Venous blood samples (3.0 mL) were collected aseptically into standard ethylenediaminetetraacetic acid (EDTA) tubes. Complete blood count (CBC) determinations were performed within 2 hours of collection using a calibrated Sysmex XP-300 automated hematology analyzer (Sysmex Corporation, Kobe, Japan). Absolute neutrophil count (ANC) and absolute lymphocyte count (ALC) were extracted to compute the NLR (ANC/ALC) [13,14].

Statistical analysis

Statistical processing was conducted using SPSS version 22.0 (IBM Corp., Armonk, NY, USA). Quantitative variables were expressed as mean ± standard deviation (SD), and categorical metrics as frequencies and percentages. Parametric two-sample comparisons utilized the independent-samples t-test, whereas multi-group comparisons were evaluated via one-way analysis of variance (ANOVA). Where exact p-values were calculated as < 0.001 by SPSS, they were reported explicitly as p < 0.001 rather than p = 0.000 to adhere to standard statistical reporting guidelines. Receiver operating characteristic (ROC) curve analysis evaluated the diagnostic capacity of NLR in distinguishing SCA cases from controls, determining the area under the curve (AUC), optimal cutoff, sensitivity, and specificity. Stratified subgroup analyses were performed across crisis types, age brackets, gender, and treatment status. Subgroup categories with limited sample sizes (such as acute chest syndrome, cerebrovascular accident, and GIT infection) were evaluated descriptively and interpreted with suitable statistical caution due to restricted power in low-frequency clinical subsets. Statistical significance was established at p < 0.05.

Ethical considerations

Ethical approval was granted by the Ethics Committee of the Gezira State Ministry of Health, Sudan. Formal written informed consent was secured from parents/guardians prior to enrollment. Participant data were anonymized to maintain confidentiality.

The study evaluated 130 participants (80 SCA cases and 50 healthy controls). Among cases, males represented 60.0% (n = 48) and females 40.0% (n = 32). The predominant age bracket was 1–5 years (36.3%, n = 29), followed by 6–10 years (32.5%, n = 26), 11–15 years (26.2%, n = 21), and > 15 years (5.0%, n = 4). A positive family history of SCA was documented in 82.5% (n = 66) of cases. Baseline disease severity stratified by hemoglobin concentration indicated severe anemia (Hb < 7 g/dL) in 48.7% (n = 39) of cases. Hydroxyurea therapy was reported in 18.8% (n = 15) of cases, whereas 90.0% (n = 72) received routine folic acid supplementation (Table 1).

Vaso-occlusive crisis (VOC) constituted the primary acute crisis event (60.0%, n = 48), followed by hemolytic crisis (12.5%, n = 10), hand-foot syndrome/dactylitis (8.8%, n = 7), acute infection (5.0%, n = 4), acute chest syndrome (2.5%, n = 2), cerebrovascular accident (2.5%, n = 2), and gastrointestinal infection (1.3%, n = 1) (Table 2). At hospital presentation, the predominant chief complaints were bone pain (42.5%, n = 34) and fever (40.0%, n = 32), followed by abdominal disturbance (10.0%, n = 8) and other symptoms (7.5%, n = 6) (Figure 1).

Figure 1: Primary clinical manifestations among pediatric SCA patients (N = 80): Bone Problems 34 (42.5%), Fever 32 (40.0%), Abdominal Disturbance 8 (10.0%), Others 6 (7.5%).

Mean NLR values were evaluated across demographic and clinical subgroups. Comparative analyses revealed that NLR did not differ significantly by sex (p = 0.616; Table 3), age group (p = 0.851; Table 4), chief presenting clinical manifestation (p = 0.121; Table 5), or underlying hemoglobin severity grading (p = 0.841; Table 7). In contrast, mean NLR differed significantly across clinical crisis categories (p = 0.001; Table 6). Patients presenting with hand-foot syndrome (dactylitis) exhibited the highest mean NLR (6.84 ± 1.66), followed by those presenting with acute infection (5.00 ± 3.06) and acute VOC (4.08 ± 2.50). Subgroup metrics for low-frequency crisis presentations (e.g., ACS, CVA, GIT infection) are listed in Table 6; however, as noted, these small subset numbers limit statistical power and necessitate cautious interpretation.

Evaluating therapeutic interventions revealed distinct biological response profiles between folic acid supplementation and hydroxyurea (HU) therapy. Folic acid administration significantly improved core red blood cells parameters—including RBC count (2.84 ± 0.74 vs. 2.05 ± 1.11 × 10^12/L, p = 0.008), Hb concentration (8.51 ± 2.23 vs. 6.20 ± 3.41 g/dL, p = 0.010), and HCT percentage (25.53 ± 6.77 vs. 18.66 ± 9.90 %, p = 0.011)—but had no statistically significant impact on mean NLR (3.70 ± 2.41 vs. 5.57 ± 3.71, p = 0.204; Table 8).

In contrast, hydroxyurea therapy was associated with a marked, statistically significant reduction in mean NLR (1.48 ± 0.33 vs. 4.44 ± 2.57 in non-treated cases, p = 0.001). Hydroxyurea also significantly improved RBC count (3.12 ± 0.70 vs. 2.68 ± 0.82 × 10^12/L, p = 0.044), Hb concentration (9.44 ± 1.96 vs. 8.01 ± 2.48 g/dL, p = 0.023), and HCT percentage (28.08 ± 5.80 vs. 24.10 ± 7.52 %, p = 0.033) (Table 9).

Pediatric SCA patients exhibited a significantly higher mean NLR compared to healthy controls (3.89 ± 2.60 vs. 1.56 ± 0.29, p < 0.001; Table 10).

Receiver operating characteristic (ROC) curve analysis evaluated the diagnostic capability of NLR to discriminate pediatric SCA patients from healthy controls. The analysis demonstrated robust diagnostic accuracy with an area under the curve (AUC) of 0.79 (95% CI: 0.71–0.87). At an optimal cut-off value of > 2.2, NLR yielded a sensitivity of 63.8% and a specificity of 98.0% (Figure 2).

Figure 2: Receiver operating characteristic (ROC) curve of NLR for differentiating pediatric SCA patients from healthy controls (AUC = 0.79, Cutoff > 2.2, Sensitivity 63.8%, Specificity 98.0%).

Sickle cell anemia (SCA) is increasingly recognized as a chronic, low-grade inflammatory state punctuated by acute, severe inflammatory surges during painful vaso-occlusive crises and infectious episodes [8,9]. In this hospital-based study from Gezira State, Sudan, pediatric patients with SCA exhibited significantly elevated mean NLR compared with healthy controls (3.89 ± 2.60 vs. 1.56 ± 0.29, p < 0.001). Furthermore, ROC curve analysis yielded an AUC of 0.79 with a specificity of 98.0% at an optimal threshold of > 2.2, supporting the clinical utility of NLR as a rapid surrogate inflammatory indicator in low-resource settings.

Pathophysiological mechanisms and cellular kinetics

The physiological mechanisms driving elevated NLR involve neutrophil priming and intravascular activation. In SCA, intravascular hemolysis releases cell-free hemoglobin and toxic heme, which stimulate endothelial adhesion molecules (P-selectin, VCAM-1) and recruit activated neutrophils. Activated neutrophils adhere to the vascular wall and directly catch rigid sickled red cells, promoting microvascular occlusion [8,18]. Neutrophils also secrete reactive oxygen species (ROS), myeloperoxidase, and inflammatory cytokines (IL-1β, IL-6, TNF-α) that amplify vascular stress. Simultaneously, stress-induced apoptosis, impaired thymic output, or splenic sequestration during acute events can induce relative lymphopenia, accelerating the rise in NLR [19]. Thus, NLR functions as a dual parameter, balancing active innate effector responses against adaptive immune status [10-12].

Comparison with regional and international literature

Our findings align closely with findings reported across sub-Saharan Africa and global cohorts. Efobi et al. in Nigeria reported significantly elevated leukocyte ratios among pediatric SCA cases during steady-state and acute crisis, identifying NLR as a robust marker of clinical disease severity [18]. Similarly, Emokpae et al. observed marked NLR elevations in patients with sickle cell nephropathy, correlating ratio magnitude with proteinuria and vascular end-organ injury [19]. In Saudi Arabia and North America, studies by Maharaj & Chang and Al-Khatti et al. confirmed that NLR spikes during acute vaso-occlusive episodes, serving as a predictor of inpatient hospital length of stay and requirement for parenteral opioid therapy [12]. Furthermore, Marchesani et al. detailed how pediatric immune cell subsets undergo dynamic shifts during acute crises, supporting our observation that NLR reflects systemic inflammatory surges [8]. In the Middle Eastern context, Zahran et al. reported parallel leukocyte dynamics in Egyptian pediatric cohorts, further substantiating NLR’s regional validity [21].

Crisis phenotypes and rationale for stratified analyses

A major finding of this study is the significant variation in mean NLR across acute clinical crisis categories (p = 0.001). Patients presenting with hand-foot syndrome (dactylitis) exhibited the highest mean NLR (6.84 ± 1.66), followed by those presenting with acute infection (5.00 ± 3.06) and acute VOC (4.08 ± 2.50). The rationale for stratifying NLR across distinct clinical crisis phenotypes stems from the differing localized and systemic inflammatory intensities characteristic of each event. Microvascular infarction within the bone marrow beds of infant hands and feet during dactylitis triggers intense localized osteonecrosis and rapid, dense neutrophil recruitment, generating extreme systemic leukocytic elevations [18,20]. In systemic infection, pathogen-driven innate immune activation drives profound neutrophil expansion. Conversely, steady-state baseline or unspecified non-acute presentation demonstrated much lower NLR levels (1.57 ± 0.50).

However, it is vital to emphasize that certain subgroup sample sizes in our study—specifically acute chest syndrome (n = 2), cerebrovascular accident (n = 2), and GIT infection (n = 1)—were small, reflecting the single-center acute admission frequency during the study period. While these subsets demonstrate elevated or fluctuating ratios, these specific subgroup numbers are insufficient for definitive statistical inferences. Therefore, subgroup-specific findings must be interpreted with caution, and future multi-center studies with larger subgroup cohorts are warranted.

Rationale for demographic and disease severity stratifications

To isolate whether NLR is driven primarily by acute inflammatory states rather than baseline demographic or hematological characteristics, we performed stratified analyses across age brackets, gender, and anemia severity. The absence of significant correlations between NLR and age (p = 0.851), sex (p = 0.616), or underlying hemoglobin grading (p = 0.841) confirms that NLR acts as a dynamic metric reflective of acute inflammatory activation rather than chronic baseline anemia or non-specific demographic differences [15-17]. This stability across age and sex brackets enhances its clinical utility in pediatric triage.

Pharmacological responses: hydroxyurea vs. folic acid

The therapeutic response profiles observed in this study provide important biological insights. Folic acid administration significantly improved core erythroid parameters (RBC count, hemoglobin, hematocrit) by supporting DNA synthesis during compensatory erythropoiesis, but had no significant effect on NLR (p = 0.204) [21]. Conversely, hydroxyurea therapy was associated with a dramatic reduction in mean NLR (1.48 ± 0.33 vs. 4.44 ± 2.57 in non-treated cases, p = 0.001). Hydroxyurea exerts potent myelosuppressive and anti-inflammatory actions by suppressing neutrophil precursor lineage proliferation, downregulating endothelial adhesion molecules (P-selectin, ICAM-1), reducing circulating reticulocytes, and elevating anti-sickling fetal hemoglobin (HbF) levels [2,21]. The near-normalization of NLR in hydroxyurea-treated pediatric cases highlights its potential utility as an accessible, cost-effective surrogate biomarker for monitoring anti-inflammatory treatment adherence and pharmacological response in resource-constrained clinics.

Clinical applicability and operational constraints

From a clinical standpoint, NLR offers substantial operational advantages in sub-Saharan African healthcare settings like Sudan. Because NLR is calculated directly from routine complete blood count (CBC) differential channels, it requires no specialized laboratory equipment or added financial costs. At a threshold of > 2.2, NLR demonstrated high specificity (98.0%), making it an effective rule-in tool to identify patients experiencing acute inflammatory spikes requiring intensified anti-inflammatory, analgesic, or antimicrobial management.

However, clinical implementation must account for important constraints. Although highly specific, the moderate sensitivity (63.8%) indicates that normal NLR levels do not entirely rule out mild or localized vaso-occlusive events. Furthermore, NLR is a non-specific inflammatory marker; concurrent subclinical bacterial/viral infections, physiological stress, corticosteroid administration, or recent blood transfusions can alter neutrophil and lymphocyte counts. Therefore, NLR should be utilized as a complementary triage and monitoring parameter alongside clinical assessment rather than a standalone diagnostic metric.

Study strengths and comprehensive limitations

A key strength of this study is providing first-hand empirical data on leukocytic inflammatory ratios among pediatric SCA patients in Sudan, detailing variations across acute crisis types and disease-modifying therapies. However, several important limitations must be acknowledged:

1. Single-Center Hospital Design and Sampling Bias: Conducting the study exclusively at Wad Medani Pediatric Teaching Hospital introduces potential sampling bias toward patients with more severe or acute clinical manifestations seeking tertiary hospital care, potentially overrepresenting elevated NLR levels compared to outpatient community steady-state populations.
2. Restricted Subgroup Sample Sizes: Subgroup sample sizes for specific acute complications (ACS n=2, CVA n=2, GIT infection n=1) were limited, restricting statistical power for robust subgroup comparisons.
3. Lack of Concomitant Inflammatory Biomarkers: Due to resource constraints in Gezira State, concurrent secondary acute-phase reactants—such as C-reactive protein (CRP), high-sensitivity CRP, ferritin, or pro-inflammatory cytokines (IL-6, TNF-a)—were not measured to correlate directly with NLR dynamics.
4. Cross-Sectional Design: The cross-sectional nature of sample collection during acute hospital presentation precludes longitudinal tracking of individual patient NLR trajectories from steady-state through acute crisis resolution.

The neutrophil-to-lymphocyte ratio (NLR) serves as a practical, low-cost inflammatory biomarker in pediatric sickle cell anemia. Because NLR is calculated directly from routine complete blood counts, it incurs no additional financial or laboratory burden. NLR effectively reflects systemic inflammatory intensity during acute vaso-occlusive episodes—particularly dactylitis and acute infection—and demonstrates a favorable pharmacological response to hydroxyurea therapy. Integrating serial NLR measurements into pediatric clinical workflows may assist clinicians in low-resource settings in evaluating acute disease activity and monitoring therapeutic response. Future prospective multi-center studies with larger subgroup cohorts and serial biomarker measurements are recommended to establish standard age-adjusted NLR reference thresholds and validate its prognostic utility in pediatric SCA management.