Pediatric Reference and Optimal Curves for Hemoglobin

Clinicians rely on reference intervals (RIs) to interpret laboratory test results. In pediatric populations, estimating RIs typically requires partitioning data by age, sex, and other relevant factors, which can lead to limited sample size and imprecise estimates; these limitations are addressed by using curve estimation, modeling hemoglobin level as a continuous function of age.

To establish hemoglobin reference curves (RCs) for children and to complement recently published World Health Organization (WHO) thresholds by estimating hemoglobin optimal curves (OCs) that may inform more appropriate reporting standards.

This cross-sectional study included healthy Canadian children aged 2 weeks through 10 years attending scheduled primary care visits from June 3, 2008, to February 26, 2020, in Toronto, Ontario, Canada. Data were analyzed from October 16, 2024, to February 1, 2026.

Blood samples were collected and analyzed for hemoglobin, ferritin, and C-reactive protein levels.

Parents completed a questionnaire to collect variables used as optimality criteria. Sex-specific RCs and OCs were estimated using nonparametric quantile regression with restricted cubic splines. RCs were based on the full sample, whereas OCs excluded children with indicators of suboptimal iron status. A web-based platform was developed to visualize these curves and calculate sex-specific reference and optimal limits by age. Findings were examined in relation to WHO hemoglobin thresholds.

Blood samples from 4597 children (2451 males [53%]; median age, 38 months [IQR, 18-63 months]) were used to estimate hemoglobin RCs, and samples from a subgroup of 3426 children (1798 males [52%]; median age, 45 months [IQR, 24-68 months]) were used to estimate OCs. For females, lower OC hemoglobin limits were slightly below the lower RC limits up until age 2 years and became higher after age 6 years (eg, at 6 months, the OC lower limit was 9.91 g/dL [90% CI, 9.70-10.13 g/dL] vs 10.00 g/dL [90% CI, 9.78-10.23 g/dL] for RC). For males, lower OC limits were higher than lower RC limits until age 20 months (eg, at 6 months, the OC lower limit was 9.74 g/dL [90% CI, 9.46-10.02 g/dL] vs 9.28 g/dL [90% CI, 8.94-9.63 g/dL] for RC) and were similar afterwards. Differences in the upper limits were minimal for both sexes. WHO hemoglobin thresholds were consistently higher than lower limits of OCs across all ages but exceeded the 5th percentile curve only among children aged 5 through 10 years for both sexes (eg, for males aged 1 year, the OC lower limit was 10.06 g/dL [90% CI, 9.92-10.21 g/dL] vs the 10.5 g/dL WHO threshold).

This cross-sectional study estimated sex-specific pediatric RCs for hemoglobin, modeled as a continuous function of age, to eliminate the need for age partitioning and overcome the associated sample size limitations. OCs, developed using health-based criteria, offered additional clinical context beyond traditional RIs. The findings highlight potential misalignments with existing WHO thresholds, particularly at younger ages.