Comparison of Anemia Screening Methods Using Paired Venous Samples in Women of Reproductive Age in Southern India.

BACKGROUND: Anemia is an important public health problem, and accurate estimates may inform policy and programs. Although hemoglobin (Hb) assessment of venous blood via automated hematology analyzers (AHAs) is recommended, most population-based surveys estimate anemia prevalence based on analysis of capillary blood via portable hemoglobinometers. OBJECTIVES: We aimed to evaluate screening methods for hemoglobin and anemia assessment using paired venous samples. METHODS: Participants were women 15-40 y who were not pregnant or lactating. Paired venous whole blood samples (n = 896) were analyzed for hemoglobin (Hb) via portable hemoglobinometer (HemoCue 301) and Coulter Counter AHA. Anemia and severe anemia were defined as Hb <12.0g/dL and <8.0 g/dL, respectively. Bland-Altman methods were used to assess the level of agreement for Hb results (mean difference, SD of differences, limits of agreement). Diagnostic accuracy parameters (sensitivity, specificity, positive predictive value, negative predictive value, accuracy) were calculated to evaluate HemoCue performance compared to the AHA reference, overall and by sociodemographic, nutritional, and metabolic characteristics. RESULTS: The estimated anemia prevalence was significantly lower via HemoCue vs. AHA (36.3% compared with 41.6%; P value < 0.0001). The HemoCue had 84.4% accuracy for anemia screening and 98.8% for severe anemia, compared to the AHA reference. The HemoCue had 74.8% sensitivity and 91.2% specificity, compared to AHA. HemoCue sensitivity was higher in women with iron deficiency [serum ferritin (SF) <15.0 µg/L: 81.6% compared with SF ≥15.0 µg/L: 41.3%], and lower in women with metabolic risk factors, including overweight [BMI ≥25.0 kg/m2: 63.9% vs. BMI <25.0 kg/m2: 78.8%], or elevated CRP (>1.0 mg/L: 67.2% vs. ≤1.0 mg/L: 82.9%), trunk fat (>35%: 62.7% vs. ≤35%: 80.1%), or whole-body fat (>35%: 63.9% vs. ≤35%: 80.3%). CONCLUSIONS: Findings suggest that women with anemia may be incorrectly identified as not anemic via portable hemoglobinometer, and anemia prevalence may be underestimated at the population level.This study was registered at clinicaltrials.gov as NCT04048330.

Estimating the serum folate concentration that corresponds to the red blood cell folate concentration threshold associated with optimal neural tube defects prevention: A population-based biomarker survey in Southern India.

BACKGROUND: RBC folate concentrations are monitored at the population level, with a recommended threshold for optimal neural tube defect (NTD) prevention. A corresponding threshold for serum folate has not been established. OBJECTIVES: This study aimed to estimate the serum folate insufficiency threshold corresponding to the RBC folate threshold for NTD prevention and examine how this threshold is modified by vitamin B12 status. METHODS: Participants were women (15-40 y; not pregnant or lactating; n = 977) from a population-based biomarker survey in Southern India. RBC folate and serum folate were measured via microbiologic assay. RBC folate deficiency (<305 nmol/L) and insufficiency (<748 nmol/L), serum vitamin B12 deficiency (<148 pmol/L) and vitamin B12 insufficiency (<221 pmol/L), elevated plasma MMA (>0.26 µmol/L), elevated plasma homocysteine (>10.0 µmol/L), and elevated HbA1c (≥6.5%) were evaluated. Bayesian linear models were used to estimate unadjusted and adjusted thresholds. RESULTS: Compared with adequate vitamin B12 status, the estimated serum folate threshold was higher in participants with serum vitamin B12 deficiency (72.5 vs. 28.1 nmol/L) or vitamin B12 insufficiency (48.7 vs. 24.3 nmol/L) and elevated MMA (55.6 vs. 25.9 nmol/L). The threshold was lower in participants with elevated HbA1c (HbA1c ≥6.5% vs. <6.5%; 21.0 vs. 40.5 nmol/L). CONCLUSIONS: The estimated serum folate threshold for optimal NTD prevention was similar to previous reports (24.3 vs. 25.6 nmol/L) among participants with sufficient vitamin B12 status. However, this threshold was more than 2-fold higher in participants with vitamin B12 deficiency and substantially higher across all indicators of insufficient vitamin B12 status (<221 pmol/L, elevated MMA, combined B12, impaired vitamin B12 status), and lower in participants with elevated HbA1c. Findings suggest a serum folate threshold for NTD prevention may be possible in some settings; however, it may not be appropriate in populations with high prevalence of vitamin B12 insufficiency. Am J Clin Nutr 2023;xx:xx-xx. This trial was registered at https://clinicaltrials.gov as NCT04048330.

A Randomized Trial of Quadruple-Fortified Salt for Anemia and Birth Defects Prevention in Southern India: Protocol Design and Methods.

Background: Women of reproductive age are at an increased risk of anemia and micronutrient deficiencies. Evidence supports the role of periconceptional nutrition in the development of neural tube defects (NTDs) and other pregnancy complications. Vitamin B12 deficiency is a risk factor for NTDs and may modify folate biomarkers that predict NTD risk at the population level. There is an interest in mandatory fortification with vitamin B12 and folic acid for anemia and birth defect prevention. However, there are limited population-representative data needed to inform policy and guidelines. Objectives: This randomized trial will be conducted to evaluate the efficacy of quadruple-fortified salt (QFS; iron, iodine, folic acid, vitamin B12) in 1,000 households in Southern India. Methods: Women 18 to 49 y who are not pregnant or lactating and reside within the catchment area of our community-based research site in Southern India will be screened and invited to participate in the trial. After informed consent, women and their households will be randomized to receive one of the following 4 interventions: 1) double-fortified salt (DFS; iron, iodine), 2) DFS + folic acid (iron, iodine, folic acid), 3) DFS + vitamin B12 (iron, iodine, vitamin B12), or 4) DFS + folic acid and vitamin B12 (QFS; iron, iodine, folic acid, vitamin B12) for 12 mo. Structured interviews will be conducted by trained nurse enumerators to collect sociodemographic, anthropometric, dietary, health, and reproductive history data. Biological samples will be collected at baseline, midpoint, and endpoint. Whole blood will be analyzed for hemoglobin using Coulter Counter. Total vitamin B12 will be measured by chemiluminescence; red blood cell folate and serum folate will be evaluated using the World Health Organization-recommended microbiologic assay. Conclusions: The results of this randomized trial will help to evaluate the efficacy of QFS to prevent anemia and micronutrient deficiencies. Clinical trial registration numbers: NCT03853304 and Clinical Trial Registry of India REF/2019/03/024479. Registration number: NCT03853304 and REF/2019/03/024479.

Iron status and inflammation in women of reproductive age: A population-based biomarker survey and clinical study.

BACKGROUND: Women of reproductive age (WRA) are at increased risk for anemia and iron deficiency. However, there is limited population-level data in India, which could help inform evidence-based recommendations and policy. AIMS: To conduct a population-based biomarker survey of anemia, iron deficiency, and inflammation in WRA in Southern India. METHODS: Participants were WRA (15-40 y) who were not pregnant or lactating. Blood samples (n = 979) were collected and analyzed for hemoglobin (Hb), serum ferritin (SF), soluble transferrin receptor (sTfR), C-reactive protein (CRP), and alpha-1 acid glycoprotein (AGP). Anemia and severe anemia were defined as Hb < 12.0 and < 8.0 g/dL. Serum ferritin was adjusted for inflammation using BRINDA methods. Iron deficiency was defined as SF <15.0 µg/L, iron insufficiency was defined as SF < 20.0 and < 25.0 µg/L, and iron deficiency anemia was defined as Hb < 12.0 g/dL and SF < 15.0 µg/L. Inflammation was defined as CRP > 5.0 mg/L or AGP > 1.0 g/L. Restricted cubic spline regression models were also used to determine if alternative SF thresholds should be used t to classify iron deficiency. RESULTS: A total of 41.5% of WRA had anemia, and 3.0% had severe anemia. Findings from spline analyses suggested a SF cut-off of < 15.0 µg/L, consistent with conventional cut-offs for iron deficiency. 46.3% of WRA had SF < 15.0 µg/L (BRINDA-adjusted: 61.5%), 55.0% had SF < 20.0 µg/L (72.7%), 61.8% had SF < 25.0 µg/L (81.0%), and 30.0% had IDA (34.5%). 17.3% of WRA had CRP > 5.0 mg/L and 22.2% had AGP > 1.0 g/L. The prevalence of ID (rural vs. urban: 49.1% vs. 34.9%; p = 0.0004), iron insufficiency (57.8% vs. 43.8%; p = 0.0005), and IDA (31.8% vs. 22.4%; p = 0.01) were significantly higher in rural areas, although CRP levels were lower and there were no differences in elevated CRP or AGP. CONCLUSIONS: The burden of anemia and iron deficiency in this population was substantial, and increased after adjusting for inflammation, suggesting potential to benefit from screening and interventions. REGISTRATION NUMBER: NCT04048330.

Anemia and Vitamin B-12 and Folate Status in Women of Reproductive Age in Southern India: Estimating Population-Based Risk of Neural Tube Defects.

BACKGROUND: Women of reproductive age (WRA) are a high-risk population for anemia and micronutrient deficiencies. However, there are few representative population-level data from India, which could help inform evidence-based recommendations and policy. OBJECTIVE: To conduct a population-based biomarker survey of anemia and vitamin B-12 and folate status in WRA as part of a periconceptional surveillance program in southern India. METHODS: Participants were WRA (15-40 y) who were not pregnant or lactating. Whole blood (n = 979) was analyzed for hemoglobin via a Coulter counter (Coulter HMX). Plasma, serum, and RBCs were processed and stored at -80°C or less until batch analysis. Vitamin B-12 concentrations were measured via chemiluminescence; RBC and serum folate concentrations were evaluated via microbiological assay. Anemia and severe anemia were defined as hemoglobin <12.0 g/dL and <8.0 g/dL, respectively. Vitamin B-12 deficiency and insufficiency were defined as total vitamin B-12 <148 pmol/L and <221 pmol/L, respectively. Folate deficiency and insufficiency were defined as RBC folate <305 nmol/L and <748 nmol/L. A previously developed Bayesian model was used to predict neural tube defect (NTD) prevalence per 10,000 births. RESULTS: A total of 41.5% of WRA had anemia and 3.0% had severe anemia. A total of 48.3% of WRA had vitamin B-12 deficiency and 74.3% had vitamin B-12 insufficiency. The prevalence of RBC folate deficiency was 7.6%, and 79.3% of WRA had RBC folate <748 nmol/L, the threshold for optimal NTD prevention. Predicted NTD prevalence per 10,000 births based on RBC folate concentrations was 20.6 (95% uncertainty interval: 16.5-25.5). CONCLUSIONS: The substantial burden of anemia, vitamin B-12 deficiency, and RBC folate insufficiency in WRA in this setting suggests an opportunity for anemia and birth defects prevention. Findings will directly inform the development of a randomized trial for anemia and birth defects prevention in southern India.This study was registered at clinicaltrials.gov as NCT04048330.

Periconceptional surveillance for prevention of anaemia and birth defects in Southern India: protocol for a biomarker survey in women of reproductive age.

INTRODUCTION: Women of reproductive age (WRA) are a high-risk population for anaemia and micronutrient deficiencies. Evidence supports the role of periconceptional nutrition in the development of adverse pregnancy complications. However, in India, there are limited population-based data to guide evidence-based recommendations and priority setting. The objective of this study is to conduct a population-based biomarker survey of anaemia and vitamin B12 and folate status in WRA as part of a periconceptional surveillance programme in Southern India. METHODS: WRA (15-40 years) who are not pregnant or lactating and reside within 50 km2 of our community research site in Southern India will be screened and invited to participate in the biomarker survey at our research facility at Arogyavaram Medical Centre. After informed consent/assent, structured interviews will be conducted by trained nurse enumerators to collect sociodemographic, dietary, anthropometry, health and reproductive history data. Venous blood samples will be collected at enrolment; whole blood will be analysed for haemoglobin. Plasma, serum and red blood cells (RBCs) will be processed and stored <-80°C until batch analysis. Vitamin B12 concentrations will be measured via chemiluminescence, and RBC and serum folate concentrations will be evaluated using the World Health Organisation (WHO)-recommended microbiological assay at our laboratory in Bangalore. A WHO surveillance system will also be established to determine the baseline prevalence of birth defects in this setting. ETHICS AND DISSEMINATION: This study has obtained clearance from the Health Ministry Screening Committee of the Indian Council of Medical Research. The study protocol was reviewed and approved by the Institutional Review Board at Cornell University and the Institutional Ethics Committees at Arogyavaram Medical Centre and St. John's Research Institute. Findings from this biomarker survey will establish the burden of anaemia and micronutrient deficiencies in WRA and directly inform a randomised trial for anaemia and birth defects prevention in Southern India. The results of this study will be disseminated at international research conferences and as published articles in peer-reviewed journals. TRIAL REGISTRATION NUMBERS: Clinical trials registration number NCT04048330, NCT03853304 and Clinical Trials Registry of India (CTRI) registration number REF/2019/03/024479.