Materials and methods

Study setting and design

This facility-based cross-sectional study was conducted at the RHTC, Barwala, North-West Delhi, India. The study was carried out over a one-year period from October 2024 to October 2025. Pregnant women attending the antenatal clinic at the RHTC during the study period were enrolled consecutively.

Study participants and eligibility criteria

The study population comprised pregnant women of any trimester attending the antenatal clinic at RHTC, Barwala. Women who provided written informed consent and were available for spot hemoglobin estimation were included in the study. Pregnant women with known hemoglobinopathies or chronic medical conditions were excluded.

Sample size and sampling technique

The sample size was calculated using the prevalence of anemia among pregnant women (86%) reported by Raj et al. [17]. Applying a 95% confidence level (Z=1.96), p=86% (0.8), q=1−p=14% (0.14), absolute precision of 5% d=0.05, and the formula, [ n=\frac{Z^{2}p(1-p)}{d^{2}} ], by substituting the values, the minimum required sample size was estimated to be 186.

A non-probability consecutive sampling technique was employed. All eligible pregnant women attending the antenatal clinic during the study period were approached consecutively and recruited until the required sample size was achieved. During the study period, 197 pregnant women were screened for eligibility. Eleven women were excluded, including seven who declined to provide written informed consent, one with a known hemoglobinopathy, and three with chronic medical conditions. The remaining 186 eligible pregnant women were enrolled and included in the final analysis.

Data collection

Data were collected using a pretested semi-structured questionnaire through face-to-face interviews. Information on sociodemographic characteristics, obstetric history, menstrual history, dietary practices, IFA supplementation, and history of worm infestation was obtained.

Dietary Assessment

Dietary intake was assessed using a single 24-hour dietary recall administered through the pretested questionnaire. Participants were asked to recall all foods and beverages consumed during the previous 24 hours. Portion sizes were estimated using standard household measures, including cups, glasses, bowls, spoons, and katoris commonly used by the participants. Daily energy (kcal), protein (g), and iron (mg) intakes were calculated using the Indian Food Composition Tables (IFCT), 2017, published by the Indian Council of Medical Research-National Institute of Nutrition (ICMR-NIN). Nutrient intakes were compared with the ICMR-NIN Recommended Dietary Allowances (RDA) for pregnant women and categorized as adequate or inadequate. The cutoffs used were 2250 kcal/day for energy, 78 g/day for protein, and 35 mg/day for iron.

Assessment of IFA Supplementation

Adequate IFA supplementation was defined as the consumption of one tablet daily containing 100 mg elemental iron and 500 μg folic acid for at least 100 days during pregnancy, in accordance with the Anemia Mukt Bharat (AMB) strategy under the National Health Mission.

Hemoglobin Estimation

Hemoglobin concentration was measured using the HemoCue® Hb 201 System (HemoCue AB, Ängelholm, Sweden). Capillary blood was collected by finger prick under aseptic precautions into a disposable HemoCue microcuvette according to the manufacturer's instructions, and hemoglobin concentration was measured immediately using the portable photometer. The HemoCue Hb 201 system measures hemoglobin using the azide methemoglobin method at wavelengths of 570 nm and 880 nm. The instrument was operated according to the manufacturer's recommendations and checked daily using the manufacturer's control cuvette. All measurements were performed by trained investigators using standardized procedures. A single hemoglobin measurement was obtained for each participant.

Anemia Classification

Anemia was classified according to the WHO criteria for pregnant women as mild (10.0-10.9 g/dL), moderate (7.0-9.9 g/dL), and severe (<7.0 g/dL)

Statistical analysis

Data were entered into Microsoft Excel (Microsoft Corp., Redmond, WA, USA) and analyzed using IBM SPSS Statistics version 25.0 (IBM Corp., Armonk, NY, USA). Continuous variables were summarized using means and standard deviations, whereas categorical variables were expressed as frequencies and percentages. Associations between anemia and categorical variables were assessed using the chi-square test. Variables that were statistically significant in the bivariate analysis and those considered clinically relevant were entered into a multivariable binary logistic regression model to identify factors independently associated with anemia. Adjusted odds ratios (AORs) with 95% confidence intervals (CIs) were reported. Statistical significance was set at p<0.05. No missing data were identified; therefore, all 186 participants were included in the final analysis.

Ethical considerations

The study was approved by the Institutional Ethics Committee of Maulana Azad Medical College, New Delhi (Ref. No. F.No.17/IEC/MAMC/2024/Comm.Med./02). Written informed consent was obtained from all participants before enrollment. The Institutional Ethics Committee-approved protocol included objectives to assess the prevalence of anemia and factors associated with anemia, in addition to evaluating the diagnostic validity of clinical pallor. Therefore, all analyses reported in this manuscript were conducted within the scope of the original ethics approval.