Regression to the mean (RTM) is a statistical phenomenon where second measurements are more likely to be closer to the mean. This is particularly observed in those with baseline values further from the mean. Anemic individuals (hemoglobin <120 g/L) are often recruited when evaluating iron supplementation programs, as they are more likely to elicit a greater hemoglobin response; however, they are also at greater risk for RTM as their baseline values are lower than the overall population mean. The aim was to calculate and apply RTM to a previously conducted iron supplementation trial of women in Cambodia at increasingly severe baseline anemia cutoffs (hemoglobin <120 g/L, <115 g/L, and <110 g/L). Women received either 60 mg/d iron (n = 191) or placebo (n = 185) for 12 wk. Hemoglobin was measured at baseline and at 12 wk (endline), and change in hemoglobin was calculated in each group for each cutoff. RTM was calculated in the placebo group at each cutoff and applied to the change observed at each cutoff in the iron group to obtain the RTM-free effect. In the placebo group, mean change in hemoglobin increased as cutoffs became more extreme (0.9 g/L to 1.9 g/L in those with baseline hemoglobin <120 g/L and <110 g/L, respectively). RTM estimates similarly increased: 1.0 g/L (<120 g/L), 1.3 g/L (<115 g/L), and 1.8 g/L (<110g/L). When applying RTM to the iron group, we found that ∼10% of the “treatment effect” could be attributable to RTM at each cutoff. However, iron supplementation was still effective in increasing hemoglobin, with an increased effect in those with lower baseline values, as proven by the RTM-free effect at each cutoff: 8.7 g/L (<120 g/L), 10.9 g/L (<115 g/L), and 13.6g/L (<110 g/L). RTM may have accounted for ∼10% of the observed change in hemoglobin following iron supplementation; however, appropriate use of a placebo group in the statistical analyses of the trial controls for this potential RTM effect.

The WHO recommends daily iron supplementation for all women in areas where the population-level anemia prevalence is ≥40%, despite the fact that hemoglobin (Hb) concentration is generally considered to be a poor prognostic indicator of iron status. In this secondary analysis, we investigated the predictive power of ten baseline hematological biomarkers towards a 12-week Hb response to iron supplementation. Data were obtained from a randomized controlled trial of daily iron supplementation in 407 nonpregnant Cambodian women (18–45 years) who received 60 mg elemental iron as ferrous sulfate for 12 weeks. Ten baseline biomarkers were included: Hb, measured with both a hematology analyzer and a HemoCue; inflammation-adjusted ferritin; soluble transferrin receptor; reticulocyte Hb; hepcidin; mean corpuscular volume; inflammation-adjusted total body iron stores (TBIS); total iron binding capacity; and transferrin saturation. Receiver operating characteristic (ROC) curves from fitted logistic regression models were used to make discrimination comparisons and variable selection methods were used to construct a multibiomarker prognostic model. Only 25% (n = 95/383) of women who completed the trial experienced a 12-week Hb response ≥10 g/L. The strongest univariate predictors of a Hb response were Hb as measured with a hematology analyzer, inflammation-adjusted ferritin, hepcidin, and inflammation-adjusted TBIS (AUC^ROC = 0.81, 0.83, 0.82, and 0.82, respectively), and the optimal cutoffs to identify women who were likely to experience a Hb response were 117 g/L, 17.3 μg/L, 1.98 nmol/L, and 1.95 mg/kg, respectively. Hb as measured with a hematology analyzer, inflammation-adjusted ferritin, and hepcidin had the best combined predictive ability (AUC^ROC=0.86). Hb measured with the HemoCue had poor discrimination ability (AUC^ROC = 0.65). Baseline Hb as measured with a hematology analyzer was as strong a predictor of Hb response to iron supplementation as inflammation-adjusted ferritin, hepcidin, and inflammation-adjusted TBIS. This is positive given that the WHO currently uses the population-level anemia prevalence to guide recommendations for untargeted iron supplementation.

Introduction The WHO recommends daily oral iron supplementation for 12 weeks in women and adolescents where anaemia prevalence is greater than 40%. However, if iron deficiency is not a major cause of anaemia, then, at best, untargeted iron supplementation is a waste of resources; at worst, it could cause harm. Further, different forms of iron with varying bioavailability may present greater risks of harm. Methods and analysis A 12-week three-arm, double-blind, randomised controlled supplementation trial was conducted in Cambodia to determine if there is potential harm associated with untargeted iron supplementation. We will recruit and randomise 480 non-pregnant women (ages 18–45 years) to receive one of three interventions: 60 mg elemental iron as ferrous sulfate (the standard, commonly used form), 18 mg ferrous bisglycinate (a highly bioavailable iron amino acid chelate) or placebo. We will measure ferritin concentrations (to evaluate non-inferiority between the two forms of iron), as well as markers of potential harm in blood and stool (faecal calprotectin, gut pathogen abundance and DNA damage) at baseline and 12 weeks. Mixed-effects generalised linear models will be used to assess the effect of iron on ferritin concentration and markers of potential harm at 12 weeks. Ethics and dissemination Ethical approval was obtained from the University of British Columbia Clinical Research Ethics Board (H18-02610), the Children's and Women's Health Centre of British Columbia Research Ethics Board (H18-02610) and the National Ethics Committee for Health Research in Cambodia (273-NECHR). Findings will be published in peer-reviewed journals, presented to stakeholders and policymakers globally and shared within participants’ communities. Trial registration number ClinicalTrials.gov Registry (NCT04017598).

Genetic hemoglobinopathies are the most common single-gene disorder worldwide. Some automated hematology analyzers have the capability of flagging individuals who may have hematological disorders based on complete blood count (CBC) biomarkers. We aimed to evaluate the accuracy of a hematology analyzer in identifying genetic hemoglobinopathies in Cambodian women and to determine which hematological biomarkers are the best predictors. A CBC was completed using a Sysmex XN-1000 analyzer and hemoglobinopathies were determined with capillary hemoglobin electrophoresis for 808 nonpregnant Cambodian women. Sysmex XN-1000 Interpretive Program (IP) messages, which flag potential hematological disorders, were produced from CBC results. Then, 2 × 2 tables were used to determine sensitivity and specificity of the IP message “Hemoglobin defect” to detect a genetic hemoglobinopathy. Receiver operating characteristic (ROC) analyses assessed the diagnostic ability of six CBC biomarkers to predict a genetic hemoglobinopathy. In total, 74% of women had a hemoglobinopathy (predominantly Hb E and α-thalassemia). “Hb defect” IP message sensitivity and specificity for genetic hemoglobinopathy detection were 10.4% and 98.6%, respectively. Variable selection strategies yielded a two-variable model including mean corpuscular volume (MCV) and red blood cell (RBC) count (AIC = 99.83, AUC^ROC = 0.98 (95% CI: 0.97, 0.99)) for the prediction of a homozygous EE disorder. Sensitivity and specificity values do not justify the use of Sysmex XN-1000 IP flag messages for identification of genetic hemoglobinopathies in Cambodian women. Development of an algorithm based on MCV and RBC biomarkers may optimize the screening ability of automated hematology analyzers.