Statistical evaluation of haematological parameters in adolescent girls supplemented with Digera muricata leaf powder: A randomized controlled design

Introduction

A historical perspective on iron deficiency anaemia research reveals a persistent methodological gap: while the biochemical consequences of iron deficiency are well characterized, the statistical rigor applied to evaluating food-based interventions has often been inadequate for generating actionable evidence. Small sample sizes, absence of control groups, single measurement timepoints, and failure to report effect sizes have collectively limited the translational impact of otherwise promising dietary iron supplementation investigations [1,2] . The repeated-measures analysis of variance (ANOVA) framework, complemented by effect size metrics such as Cohen's d and partial eta-squared, provides a statistically appropriate and clinically interpretable approach for evaluating interventions with multiple measurement timepoints. This framework allows for partitioning of variance into Group, Time, and Group x Time interaction components, with the interaction effect indicating differential trajectories of change between groups representing the critical test of intervention efficacy [3] . Reporting of effect sizes beyond p-values is increasingly recognized as essential for quantifying clinical relevance and enabling meaningful meta-analytic synthesis across investigations [4] .

This research was specifically designed to address this statistical evidence gap by applying a pre-test/post-test randomized controlled design with: (i) adequate sample size calculated a priori using power analysis; (ii) three measurement timepoints (baseline, Day 45, Day 90) enabling assessment of temporal trajectory as well as final outcome; (iii) repeated-measures ANOVA with Bonferroni correction for all primary outcomes; (iv) Cohen's d calculation for each parameter at each timepoint; and (v) partial eta-squared reporting to contextualize the practical significance of observed effects.

Materials and Methods

Materials

Methods

Ninety eligible girls were randomized using computergenerated block randomization (block size 6) into supplement (n=45) or placebo control (n=45) groups. Supplement group participants received one sachet of 10 g D. muricata leaf powder per day to be dissolved in a standardized cup of warm water and consumed after the morning meal; placebo group received an identically presented 10 g cornstarch sachet. Both groups received equivalent dietary counselling and were asked not to alter other dietary habits. Haematological assessments (haemoglobin by Sysmex haematology analyzer, serum ferritin and serum iron by ELISA, TIBC by colorimetric method, transferrin saturation calculated as serum iron/TIBC x 100) were performed at baseline, Day 45, and Day 90. Data were entered in duplicate into SPSS v27.0 and checked for transcription errors. Repeated-measures ANOVA with Bonferroni post-hoc correction was applied for all primary endpoints. Cohen's d was calculated as (mean1 -mean2) / pooled SD; partial eta-squared was extracted from the ANOVA output. A priori power analysis (G*Power 3.1) specified a medium-to-large Group x Time interaction effect (f=0.30, alpha=0.05, power=0.80, 3 timepoints, correlation among measurements=0.6), yielding a minimum requirement of 42 participants per group; 45 were enrolled per group to allow for up to 7% attrition. Statistical significance was set at p <0.05.

Results

Participant Flow and Baseline Characteristics

Of 118 girls screened, 90 met eligibility criteria and were enrolled (supplement n=45, control n=45). Three participants withdrew before Day 45 (supplement n=1, control n=2; reasons: relocation n=2, personal n=1), leaving 87 participants who completed the full 90-day protocol. Intent-to-treat analysis with last observation carried forward (LOCF) was applied for the three withdrawals. Baseline haematological parameters did not differ significantly between groups (independent t-test, all p>0.05), confirming successful randomization. Baseline characteristics: supplement group mean age 14.8±1.2 years, mean Hb 9.82±0.62 g/dL, mean serum ferritin 14.2±4.8 ng/mL; control group mean age 15.0±1.1 years, mean Hb 9.76±0.64 g/dL, mean serum ferritin 14.6±4.6 ng/mL.

Discussion

The strong Group x Time interaction effects observed across all haematological parameters (partial eta-squared 0.176-0.246, all p <0.001) provide statistically robust evidence of the efficacy of D. muricata leaf powder supplementation at 10 g/day for 90 days in iron-deficient adolescent girls. The comparison with existing literature is informative: a metaanalysis of food-based iron interventions in adolescent girls across 14 randomized trials reported pooled effect sizes for haemoglobin of Cohen's d=0.64 (95% CI: 0.42-0.86) [8] , compared to the d=1.42 observed in the current research at Day 90, suggesting that D. muricata leaf powder may deliver superior haematological outcomes relative to other evaluated food-based iron sources, though direct head-to-head comparisons are required to confirm this inference.

The temporal trajectory of effect sizes from medium at Day 45 (d=0.44-0.52) to large at Day 90 (d=0.82-1.68) is consistent with the physiological kinetics of haematological recovery. Haemoglobin regeneration requires iron repletion of bone marrow stores before erythropoietic acceleration can be fully expressed, a process that typically requires 6-8 weeks in moderately iron-deficient individuals [9] . The accelerating effect size trajectory observed between Day 45 and Day 90 is therefore mechanistically coherent and suggests that extending the supplementation period beyond 90 days might yield further incremental improvements, particularly for serum ferritin, which serves as the primary indicator of iron store replenishment rather than functional iron status [10] .

The partial eta-squared values (0.176-0.246) classify the Group x Time interaction effects as medium-to-large by the conventional thresholds (0.01 small, 0.06 medium, 0.14 large) established for this metric [11] . This contextualizes the statistical significance in terms of practical importance: the observed Group x Time interaction explains between 17.6% and 24.6% of the total variance in haematological trajectories, which represents a substantial treatment-attributable component given the heterogeneity typical of clinical nutritional investigations. Agreement with this interpretation is found in comparably rigorous randomized designs evaluating iron-enriched bread [12] and legume-based iron supplements [13] in adolescent populations, where partial etasquared values of 0.12-0.22 have been reported for haematological outcomes.

Statistical methodology note

The choice of repeated-measures ANOVA over independentsamples comparisons at each timepoint was deliberate and methodologically appropriate for this research design. Repeated-measures designs exploit within-subject correlations across timepoints, yielding greater statistical power for detecting Group x Time interaction effects than crosssectional comparisons while controlling for individual baseline differences [14] . The confirmation of sphericity (Mauchly's W>0.90 for all parameters) validated the use of uncorrected degrees of freedom; where sphericity is violated, Greenhouse-Geisser or Huynh-Feldt correction should be applied, and future reports of similar designs should explicitly report Mauchly's test results as part of the statistical methods documentation [15] .

Conclusion

This research establishes, through rigorous randomized controlled design and full statistical reporting including repeated-measures ANOVA, partial eta-squared, and Cohen's d, that Digera muricata leaf powder supplementation at 10 g/day for 90 days produces clinically meaningful and statistically large-effect improvements in haemoglobin (d=1.42), serum ferritin (d=1.68), serum iron (d=1.28), TIBC (d=0.82), and transferrin saturation (d=1.14) in iron-deficient adolescent girls. These findings strengthen the evidence base for food-based iron interventions using D. muricata and provide the statistical parameters effect sizes and partial etasquared values needed for power calculation in future investigations and meta-analytic synthesis. Future research should investigate minimum effective supplementation periods, optimal dose-response relationships, and comparative efficacy against pharmacological iron-folic acid supplementation in larger, multi-site randomized trials.

Acknowledgement

AcknowledgementsInstitutional support for this research was provided by the

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