Module 3. Biomarker selection and specimen handling

Micronutrient and related health indicators

Vitamin A

Anaemia

Iron deficiency

Iron deficiency anemia

Iodine

Vitamin B12

Folate

Vitamin D

Zinc

Overview of specimen collection containers, storage and transport conditions

Introduction

Many factors drive the selection of indicators. Some of these factors relate to participants, for example the acceptability and feasibility of the collection methods. Others concern field work (the ease of specimen collection, processing, storage, and transportation) and laboratory issues such as equipment, training, reagents, and costs. All these factors must be considered during the planning stage of the survey.

This module provides information to guide the selection of appropriate and reliable biomarkers, types of specimens, and methods of collection and analysis applicable to large micronutrient surveys. For each micronutrient there is a discussion of: specimen collection and management, biomarker analysis, an approximate budget, and how to interpret results. Issues of quality control are described where appropriate. The recommended collection containers, processing, storage and transport conditions for different specimen types are summarized in Table 3.11 at the end of the module. Costs have been calculated based on prices known in 2019. Actual costs for analyses may differ greatly from one laboratory to another.

Advice about the most suitable indicators and methods change periodically. During the planning phase, it is advisable to review the latest recommendations and consult with appropriate experts. Review papers on micronutrient status assessment developed under the Biomarkers of Nutrition for Development (BOND) program are shown in Box 3.1.

Many factors drive the selection of indicators. Some of these factors relate to participants, for example the acceptability and feasibility of the collection methods. Others concern field work (the ease of specimen collection, processing, storage, and transportation) and laboratory issues such as equipment, training, reagents, and costs. All these factors must be considered during the planning stage of the survey.

This module provides information to guide the selection of appropriate and reliable biomarkers, types of specimens, and methods of collection and analysis applicable to large micronutrient surveys. For each micronutrient there is a discussion of: specimen collection and management, biomarker analysis, an approximate budget, and how to interpret results. Issues of quality control are described where appropriate. The recommended collection containers, processing, storage and transport conditions for different specimen types are summarized in Table 3.11 at the end of the module. Costs have been calculated based on prices known in 2019. Actual costs for analyses may differ greatly from one laboratory to another.

Advice about the most suitable indicators and methods change periodically. During the planning phase, it is advisable to review the latest recommendations and consult with appropriate experts. Review papers on micronutrient status assessment developed under the Biomarkers of Nutrition for Development (BOND) program are shown in Box 3.1.

Iron deficiency anaemia

Individuals with iron deficiency anaemia are anaemic (based on low haemoglobin) and iron deficient (based on one of the recommended indicators of iron status). Fig. 3.1. shows two examples of the relationship between iron deficiency, iron deficiency anaemia and anaemia in populations with the same prevalence of anaemia. The level of overlap between these three conditions will vary between populations, and within a given population. In setting A, the prevalence of iron deficiency in the population is high, as is the prevalence of iron deficiency anaemia, while non-iron deficiency causes of anaemia are infrequent. In setting B, the prevalence of iron deficiency and iron deficiency anaemia are lower than in setting A, indicating that other factors, such as malaria, hookworm, and/or genetic blood disorders (for example, haemoglobinopathies) may be the main causes of anaemia.

Fig. 3.1. Visual representation of the overlap between iron deficiency, iron deficiency anaemia and anaemia

Adapted from: reference ¹.

Vitamin D

Vitamin D is produced endogenously when ultraviolet-B (UVB) rays from sunlight strike the skin and trigger vitamin D synthesis. It can also be obtained from the diet in the form of vitamin D3, cholecalciferol, from animal sources (e.g. fatty fishes such as salmon, tuna and mackerel, fish liver oils, beef liver, cheese and egg yolks) or vitamin D2, ergocalciferol, in mushrooms irradiated with UVB light, vitamin D-fortified foods (dairy products, oils, margarine and spreads, and some breakfast cereals) and vitamin D-containing supplements ^1,2. Vitamins D2 and D3 are similar compounds except for the structure of their side chains. The conversion of vitamins D2 and D3 into active compounds requires a two-step enzymatic hydroxylation process, although they have different conversion efficacy ³. A meta-analysis indicates supplementation with vitamin D3 had a significant and positive effect in raising serum 1,25-dihydroxyvitamin D (1,25(OH)2D) concentrations, the physiologically active form also known as calcitriol, compared to supplementation with vitamin D2 (P = 0.001) ⁴. However, vitamin D2 is considered an active substance and is not excluded as a source of dietary vitamin D.

The most widely accepted and used indicator of vitamin D status is plasma or serum 25-hydroxyvitamin D (25(OH)D), which is reflective of exposure to vitamin D from both cutaneous synthesis and dietary intake from food and supplements ⁵. However, there is no international consensus about the blood concentration associated with optimal status in different population groups ⁶ and WHO has not yet issued guidance. The United States’ Institute of Medicine (IOM) of the National Academies (now referred to as the Health and Medicine Division of the National Academies of Sciences, Engineering, and Medicine [the National Academies]) established vitamin D recommended nutrient requirements for populations based on preventing serum 25(OH)D concentrations below 30 nmol/L for musculoskeletal outcomes ⁷. The Endocrine Society, a global organization representing professionals from the field of endocrinology, defines vitamin D deficiency as 25(OH)D concentrations below 50 nmol/L (20 ng/mL) and vitamin D insufficiency as 52.0–72.5 nmol/L (21–29 ng/mL), based on multiple health outcomes, including but not limited to musculoskeletal outcomes ⁸. The European Food Safety Authority (EFSA) has suggested similar cutoff values ⁹.

Specimen collection and management: 25-hydroxyvitamin D is usually measured in plasma or serum specimens obtained by centrifugation of whole blood collected by venipuncture. 25-hydroxyvitamin D is very stable, so whole blood processing can be delayed for up to two days. Serum is stable for at least two weeks at 4˚C and for at least one year at -20˚C ¹⁰.

Biomarker analysis: Serum 25-hydroxyvitamin D is commonly measured by a competitive protein-binding assay on a fully automated clinical analyser using commercial assay kits, or by using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The latter approach is resource intensive and not suited for laboratories with limited capacity and infrastructure. The required analysis volume is typically >100 µL, although a minimum specimen volume of >300 µL is needed for repeat analysis. The product sheet for the intended assay will specify the specimen matrix requirements and should be consulted before deciding on the method and ordering survey supplies. Serum is the preferred matrix, since not all assays can utilize EDTA or heparin plasma. Appropriate quality control measures must be followed to ensure high quality results. The assay kits include calibration materials and often also include quality control materials. It is nonetheless recommended to establish “in-house” quality control materials that can be tracked over a longer period to verify that the method did not shift over time. The method imprecision is typically 5–10%.

Serum-based certified reference materials are available from NIST (SRM 972a) to verify method accuracy. However, not every assay may be able to use this material because the assay performance may differ between native patient samples and reference materials that have undergone some processing. Because of the variations in results between assays and between laboratories, efforts have been made to improve assay standardization. The United States’ Office of Dietary Supplements of the National Institutes of Health established the Vitamin D Standardization Program to improve the standardization of 25(OH)D assays. Additionally, the United States Centers for Disease Control Vitamin D Standardization Certification Program (VDSCP) provides participating laboratories with one-time sets of 40 different reference materials for bias assessment and calibration, as well as 40 blinded samples per year with assigned values measured by a reference LC-MS/MS method for both 25(OH)D2 and 25(OH)D3, to certify analytical performance such as bias and imprecision ¹¹. Over 20 laboratories and assay manufacturers are currently participating in the CDC programme ¹². Additionally, the Vitamin D External Quality Assessment Scheme, from the Charing Cross Hospital, UK, provides participating laboratories with 20 samples per year that have reference values for 25(OH)D2 and 25(OH)D3, for assessment of bias and to allow for inter-assay and between-laboratory comparisons ¹³.

Approximate budget requirements for analysis: The cost for a clinical analyser can vary widely but is typically around US$ 100 000. The cost for materials and supplies is approximately US$ 10–20 per sample.

Interpretation of results: There are no WHO recommended cutoff values for defining risk of deficiency at the individual or the population level. The United States IOM of the National Academies identified serum 25(OH)D concentrations for determining vitamin D status at the individual level in all age groups ⁷ (see Table 3.9). These values are similar to those proposed by a group of experts for the context of skeletal mineralization and mineral ion metabolism for the prevention of nutritional rickets ¹⁴.

Table 3.9. 25-Hydroxyvitamin D concentrations in serum for determining individual level vitamin D status in all age groups^a

Serum levels (nmol/L)	Interpretation
<30	Risk of deficiency
30-<50	Risk of insufficiency
50-75	Likelihood of sufficiency
>75-125	No increased benefit
>125	Risk of excess

^a Source: reference ⁷.

Iodine

Iodine is necessary for proper foetal brain development, and there is mixed evidence that high levels of iodine may affect thyroid function ¹. The most usual indicator of population iodine status is the median urinary iodine concentration (UIC) of the population.

There are clinical and other biological indicators of iodine status, including goitre and thyroid volume by ultrasonography, however these are not currently recommended for large national cross-sectional surveys. Assessment of goitre is not recommended either, because it is a more subjective method, especially if conducted by palpation. More importantly, thyroid size responds very slowly after the introduction of an intervention (such as iodized salt) to improve iodine intake, and does not provide a reliable indication of recent individual or population iodine intake ¹.

More recently, thyroglobulin has been proposed as a sensitive indicator of both low and excess iodine intake for use at the population level, following a U‐shaped association with the urinary iodine concentration (UIC) in school-age children, pregnant women and infants. Due to high day‐to‐day variability, however, the utility of thyroglobulin as an individual biomarker of iodine status is uncertain ².

Urinary iodine concentration (UIC):

The recent iodine intake of an individual can be assessed by measuring urinary iodine excretion (UIE), the iodine level in a 24-hour collection of urine that mitigates diurnal variations in iodine excretion. However, it is not feasible to include 24-hour urine collections as part of a large cross-sectional survey, and thus it is not possible to estimate the iodine status of individual survey participants. With a sufficiently large number of single urine samples, the median UIC will represent the status of the entire sample population.

Specimen collection and management: Urine samples are normally collected at the household but depending on the survey they can be collected from a clinic or laboratory. Samples do not require refrigeration but are usually kept in cool boxes or refrigerated with other specimens until they are processed.

Urine samples are relatively easy to collect from older children and adults. Only a small amount of urine is required (approximately 1 mL) for duplicate laboratory testing of iodine content. Infants and very young children may have difficulty urinating on demand or into the collection cup.

Biomarker analysis: The urinary iodine ammonium persulfate method ³ is considered the “gold standard” assay for analysis of UIC. Reagents and calibration materials, including ammonium persulfate, arsenious acid, ceric ammonium sulphate solutions and sulfuric acid are necessary for the sample analysis. A sample of 250 µL is needed, but a minimum of 1 mL of urine should be collected for potential repeat analysis. It is recommended to use internal quality controls during each analytical run in order to assess the accuracy and precision of the results. CDC also offers the Ensuring the Quality of Urinary Iodine Procedures (EQUIP) programme for urinary iodine, available globally to all laboratories so that bias and imprecision of their method can be tested against the CDC method three times a year ⁴. The urinary iodine ammonium persulfate method is relatively simple to perform but requires special attention to prevent iodine contamination of the laboratory area and equipment. Although urinary iodine excretion can be expressed in relation to creatinine excretion (µg iodine/g creatinine), the ratio of urinary iodine to creatinine can be misleading ⁵. Therefore, WHO recommends reporting urinary iodine as µg/L ⁶.

Approximate budget requirements for analysis: Instrumentation needed for this method includes a spectrophotometer, laboratory glassware, regents, a vortex, a heating block with a timer, and various pipettes (costing approximately US$ 10 000). The cost for materials and supplies is approximately US$ 2 - 5 per sample.

Interpretation of results: Table 3.6 presents the median UIC that indicates iodine status among different population groups ⁶. It is important to note that only population-level assessments of iodine status are possible from the survey methodology of casual, spot urine sample collection. Iodine status estimates based on the methodology of casual spot urine sample collection cannot be used to classify individual status and should not be presented as a prevalence of deficiency or adequacy ¹. The information provided in Table 3.6 is frequently misinterpreted to reflect the situation of individuals. The correct interpretation is that populations with a median urinary iodine <20 μg/L have “severe” iodine deficiency, populations with a median urinary iodine 20-49 μg/L have “moderate” iodine deficiency, and populations with a median urinary iodine 50-99 μg/L have “mild” iodine deficiency.

Table 3.6. Epidemiologic criteria for assessing population-level iodine nutrition based on median urinary iodine concentrations in different population groups^a

Median urinary iodine concentration (μg/L)	Indication of population iodine intake	Indication of population iodine status
School-age children (6-12 years of age)
<20	Insufficient	Severe iodine deficiency
20-49	Insufficient	Moderate iodine deficiency
50-99	Insufficient	Mild iodine deficiency
100-199b	Adequate	Adequate iodine nutrition
200-299	Above requirements	May pose a slight risk of more than adequate iodine intake in these populations
≥300	Excessivec	Risk of adverse health consequences (iodine-induced hyperthyroidism, autoimmune thyroid disease)
Pregnant women
<150	Insufficient
150-249	Adequate
250-499	Above requirements
≥500	Excessivec
Lactating women and children under 2 years of age
≥500	Insufficient
≥100	Adequate

^a Source: reference ⁶

^b A UIC range of 100-299 µg/L has been proposed to indicate optimal iodine status among school age children^1,7.

^c The term “excessive” means in excess of the amount required to prevent and control iodine deficiency.

⁷

Overview of specimen collection containers, storage and transport conditions

Table 3.11 presents a summary of the collection device requirements for different specimen types and main factors related to specimen storage and transport.

Biological matrix and collection method	Collection, testing and storage device/container	Specimen type	Biomarker(s)	Storage and transport considerations
Capillary blood from a finger prick	Microcuvette for portable photometer (tested in field)	Whole blood	Haemoglobina	Test immediately, ensure that cuvettes are kept in dry and air tight container. Do not expose un-used cuvettes to the air.
	Microcuvette for portable photometer (tested in field)	Whole blood	Haemoglobina, folate (RBC)	Test immediately or refrigerate specimen and test (Hb) or process specimen (RBC folate) within 1-2 days of blood collection.
		Plasma (after centrifugation)	Ferritin, sTfR, RBP, AGP, CRP	Refrigerate whole blood and generate plasma within 2-3 days of blood collection. Plasma stable for 1 week refrigerated and 1 year at -20˚C. Ship frozen samples on dry ice.
	Small blood collection tube without anticoagulant	Serumb	Ferritin, sTfR, RBP, AGP, CRP	Refrigerate whole blood and generate serum within 1 day of blood collection. Serum stable for 1 week refrigerated and 1 year at -20˚C; ship frozen samples on dry ice.
Venous blood from venipuncture	Vacuum tube with EDTA	Whole blood	Haemoglobina folate (RBC), B12	Refrigerate and protect whole blood from light and generate haemolysate within 1-2 days of blood collection. Haemolysate is stable for 1 month at -20˚C; long-term storage at -70˚C; ship frozen samples on dry ice.
		Plasma (after centrifugation)	Ferritin, sTfR, RBP, AGP, CRP, retinol, MRDR	Refrigerate whole blood and generate plasma within 2-3 days of blood collection. Plasma stable for 1 week refrigerated and 1 year at -20˚C. Ship frozen samples on dry ice.
		Dried blood spot (RBC folate)	Folate (RBC)	Refrigerate and protect whole blood from light and generate DBS within 1 day of blood collection. Dry DBS at ambient temperature; DBS stable for 2 days refrigerated and 1 year at -20˚C. Long-term storage at -70˚C. Ship frozen samples on dry ice.
	Vacuum tube without anticoagulant and with a serum separator – trace metal freeb	Serum (after centrifugation)	Zinc, ferritin, sTfR, RBP, AGP, CRP, retinol, MRDR, folate (serum), vitamin B12 & vitamin D (25OHD)	Refrigerate whole blood and generate serum within 1-2 days of blood collection. Serum stable for 1 week refrigerated and 1 year at -20˚C. Ship frozen samples on dry ice.
Urine sample	Sterile cup with lid (could be transferred to sterile cryogenic vial)	Urine	Urinary iodine	Can be stored refrigerated or frozen at -20˚C. Ship frozen samples on dry ice.

EDTA: ethylenediaminetetraacetic acid; sTfR: soluble transferrin receptor; RBC: red blood cell; RBP: retinol binding protein; AGP: alpha-1-acid glycoprotein; CRP: C-reactive protein; MRDR: modified relative dose response.

^a Whole blood can be collected into microcuvette directly from finger or heel prick or from whole blood collected in a small blood collection tube or vacuum tube.

^b Trace metal free tubes needed for zinc assessment.

Zinc

Serum zinc is the key indicator for assessing zinc deficiency among populations. Serum zinc reflects dietary zinc intake and responds consistently to zinc supplementation. Reference data are available for most age and sex groups. Assessing serum zinc allows the identification of specific populations and subgroups who have an elevated risk of zinc deficiency ¹. Serum zinc cannot be used in the diagnosis and treatment of individuals who are zinc deficient because serum zinc concentrations are not a direct reflection of the individual’s zinc status ². However, serum zinc can provide an assessment of the magnitude of the risk of zinc deficiency within a specific population or subgroup ¹.

There are special considerations when measuring serum zinc concentration. Recent meals, the time of day, the age and sex of the participant, the use of hormonal contraception, inflammation and systemic infections all can have a direct impact on serum zinc concentration ². The International Zinc Nutrition Consultative Group (IZiNCG) suggests collecting information on inflammatory proteins when assessing zinc, and if there is a significant negative correlation between either inflammatory protein and serum zinc, to adjust serum zinc concentration statistically ³.

Zinc concentrations in hair, nail and urine have been used in some studies to assess a population’s exposure to zinc; however, they are currently not recommended as a single indicator in the assessment of zinc status ^4,5.

Specimen collection and management: There are special considerations when collecting serum specimens to assess zinc deficiency. Specimens can be easily contaminated by ambient sources of zinc during collection, processing and analysis. To reduce the risk of ambient zinc contamination, the blood specimen must be taken directly from the vein using zinc-free needles and trace-metal-free vacuum blood collection tubes. When processing the samples, zinc-free sterile storage vials and transfer pipettes or pipette tips must be used to reduce the risk of contamination. If possible, specimens should be collected from survey participants during a pre-agreed time of day, and the participants should have been fasting for at least eight hours prior to collection. Fasting conditions are not always possible, so if non-fasting specimens are collected, an effort must be made to reduce variation by collecting samples from the entire survey population during the same time of day. It is important to record the time of the previous meal, especially for calculating non-fasting cutoff values and interpreting data. Once a blood specimen has been collected, it should be stored in a cold box or a refrigerator until centrifuged to collect the serum.

Ideally, all specimens should be centrifuged within 20 to 30 minutes after collection. If this is not possible, it is recommended that processing be done within 24 hours, provided that the cold chain is reliable and blood specimens can be kept refrigerated (2–10°C). Specimens should also be centrifuged in a controlled laboratory environment and serum should be separated into vials under a laboratory hood. In situations where this is not feasible, it is recommended that a tent be used so that a sealed laboratory can be configured. Plastic boxes can be used along with plexiglass or plastic wrap to develop a temporary field hood. All necessary information specific to zinc-free laboratory supplies and the construction of a hood from a plastic box is available from IZiNCG.

Biomarker analysis: Zinc concentration can be measured using several different analytical methodologies. The gold standard is inductively coupled plasma mass spectrometry (ICP-MS), however, this method is very expensive. Other acceptable analytical methods to measure zinc concentration include inductively coupled plasma atomic emission spectrometry, flame atomic absorption spectrometry (AAS), graphite furnace atomic absorption spectrometry, and neuron activation analysis.

Approximate budget requirements for analysis: The cost for instrumentation can vary widely depending on use of AAS or ICP-MS, but is typically around US$ 50 000–US$ 400 000. The cost for materials and supplies is approximately US$ 10–20 per sample.

Interpretation of results: Data on low serum zinc concentration should be compared with the appropriate reference data for age, sex, time of day and time since last meal. Reference data have been derived from NHANES II and represent individuals older than 2 years of age. The IZiNCG-recommended cutoff values for low serum zinc concentration are presented in Table 3.10. It is important to note that if 20% of the population has low serum zinc concentration there is a risk for zinc deficiency and a public health concern ².

Table 3.10. Lower cutoff values for serum zinc concentration by age group, sex, time of day and time since last meal, recommended by the International Zinc Nutrition Consultative Group (IZiNCG) ^a

	Suggested lower cutoff values for low serum zinc concentration in µg/dL (µmol/L)b
Time of day and fasting status	≤10 years	≥10 years
	Males and females	Non-pregnant females	Males
Morning, fastingc	not available	70 (10.7)	>74 (11.3)
Morning, non-fasting	65 (9.9)	66 (10.1)	70 (10.7)
Afternoon, non-fasting	57 (8.7)	59 (8.6)	61 (9.3)

^a Source: reference ⁵.

^b Values converted to µmol/L by dividing µg/dL by 6.54.

^c Fasting is defined as no food or beverage consumption for at least 8 hours.

Vitamin B12

Vitamin B12 is found in animal source foods. A chronic dietary deficiency of vitamin B12 contributes to failure to thrive in infants and to neurologic disorders among all age groups. It is one nutrient deficiency that causes macrocytic anaemia. Although strict dietary deficiencies are rare among populations consuming a Western diet, some population groups consume minimal or no animal source foods due to abject poverty, to religion or to other customs ^{1, 2}. More common amongst the elderly, vitamin B12 deficiency may also result from inability to absorb vitamin B12 based on an underlying disorder of the stomach or intestine, such as hypertrophy of the intestines, reduced gastric acidity, lack of intrinsic factor, or an interference with medications. The autoimmune condition known as pernicious anaemia, most commonly experienced by elderly populations, is a rare but important disease that inhibits absorption of vitamin B12 and will result in deficiency if untreated ³.

The most commonly used biomarker to assess vitamin B12 status in population-based surveys is serum vitamin B12 (total cobalamin) ³.

Specimen collection and management: Serum samples are obtained by centrifugation from whole blood collected by venipuncture. The whole blood needs to be protected from light and processed to serum within a few days of blood collection. Serum vitamin B12 is stable for at least one week at 4˚C, and for at least one year at -20˚C ⁴.

Biomarker analysis: Serum vitamin B12 is commonly measured via a competitive protein-binding assay on a fully-automated clinical analyser using commercial assay kits. The required analysis volume is typically around 25 µL, however a minimum specimen volume of 150 µL may be needed to fill the sample cup for the clinical analyser. The product sheet for the intended assay will specify the specimen matrix requirements and should be consulted before deciding on the method and ordering survey supplies. Serum is the preferred matrix, since not all assays can utilize EDTA or heparin plasma. Appropriate quality control measures must be followed to ensure high quality results. The assay kits include calibration materials, and in many cases they also include quality control materials. It is nonetheless recommended to establish “in-house” quality control materials that can be tracked over a longer period to verify that the method did not shift over time. The method imprecision is typically 5–10%. A WHO developed serum-based international standard (IS 03/178) is available through the NIBSC to verify method accuracy. However, not every assay may be able to use this material because the assay performance may differ between native patient samples and reference materials that have undergone some processing. Moderate assay differences can be observed in proficiency testing programmes, such as the US College of American Pathologists (CAP) Ligand Survey ⁵. CDC’s Performance Verification Program for Serum Micronutrients ⁶ covers serum vitamin B12 and CDC also offers quality control materials for this analyte to support in-house quality assurance programmes for laboratories engaged in public health work ⁷.

Approximate budget requirements for analysis: The cost for a clinical analyser can vary widely but is usually around US$ 100 000. The cost for materials and supplies is approximately US$ 3–5 per sample.

Interpretation of results: To estimate vitamin B12 deficiency at the individual level, the WHO recommended cutoff value is 203 pg/mL (150 pmol/L) ⁸. This is the point where the slope of the relationship between serum vitamin B12 and methylmalonic acid changes and where serum methylmalonic acid concentrations rise steeply in response to decreasing serum vitamin B12 concentrations. This is nearly identical to the clinically derived cutoff value of 200 pg/mL (148 pmol/L), below which there are often metabolic abnormalities present. Serum vitamin B12 concentrations between 200 and 300 pg/mL are frequently characterized as “subclinical” deficiency, or at risk of deficiency (depletion), but it is less clear whether these concentrations have a negative health impact ^{1, 8}.

Iron deficiency

Recommended indicators of iron status for determining iron deficiency include serum ferritin, serum soluble transferrin receptor, and total body iron ^1,2.

Ferritin

Serum ferritin is the most specific, non-invasive biochemical test to quantify total body iron stores. In the absence of inflammation, the concentration of serum ferritin is positively correlated with the size of the total body irons stores, with a low serum ferritin concentration reflecting depleted iron stores and therefore iron deficiency ³. However, serum ferritin is an APP and is elevated in response to infectious or inflammatory processes. In population- based surveys, this may artificially lower the prevalence of iron deficiency, thus It is recommended that serum ferritin be assessed along with measures of inflammation (e.g. CRP and/or AGP).

There are several methods to account for the increase in ferritin values caused by inflammation. One method is to adjust serum ferritin concentrations based on CRP and AGP using regression or arithmetic correction factors, such as the BRINDA ⁴ or Thurnham methods ⁵, respectively. When using these correction approaches to adjust ferritin concentrations, you can apply the cutoff values recommended for apparently healthy populations. Another method is to apply a higher serum ferritin cutoff value that defines deficiency, 30 μg/L or 70 μg/L, depending on the age group, to individuals with infection or inflammation ³.

Specimen collection and management: Most commonly, ferritin is measured in serum or plasma samples that are obtained by centrifugation from whole blood collected by venipuncture or finger prick. The whole blood needs to be refrigerated as soon as possible and processed to serum or plasma within 48 hours of blood collection. Ferritin in serum is stable for at least one week at 4˚C and for at least one year at -20˚C ⁶.

Biomarker analysis: Ferritin is measured using immunoassays, including methods that can be conducted either with a fully-automated clinical analyser or a manually executed ELISA assay. Commercial assay kits are available for both types of assay. There is no significant difference in within-run imprecision, between-run imprecision, limit of detection, recovery rate or linearity between commercial and home-made assays, or between automated multiple-analytes detection equipment and single laboratory apparatus, showing, overall, that the most common methods used for ferritin determinations are comparable ⁷.

The required analysis volume is typically <25 µL serum, however a minimum specimen volume of >150 µL serum may be needed to fill the sample cup for the clinical analyser. The product sheet for the intended assay will specify the specimen matrix requirements and should be consulted before deciding on the method and ordering survey supplies. Serum is the preferred matrix, since not all assays can use EDTA or heparin plasma. Sandwich ELISAs are also available to measure serum ferritin along with other indicators, including those assessing other iron indicators and vitamin A and inflammation status ⁸.

Quality control measures are required to ensure high quality results ⁹. Commercial assay kits include calibration materials and may also include quality control materials. It is nonetheless recommended to establish “in-house” quality control materials that can be tracked over a longer period to verify that the method did not shift over time. The method imprecision is typically 5–10% for clinical analyser assays and around 10% for ELISA assays. Ferritin is also part of the CDC VITAL-EQA program and of CDC’s Performance Verification Program for Serum Micronutrients ¹⁰.

A WHO-developed serum-based international standard (recombinant ferritin RM 94/572) ¹¹, used to verify method accuracy, is available through the National Institute of Biological Standards and Control (NIBSC). However, not every assay may be able to use this material because the assay performance may differ between native patient samples and reference materials that have undergone some processing. Quality control materials for serum micronutrients including ferritin are available from CDC to support in-house quality assurance programs for laboratories engaged in public health work ¹². Moderate assay differences (e.g. 2.0–5.0% variability depending on the assay) are common in proficiency testing programmes, such as the US College of American Pathologists (CAP) Chemistry Survey ¹³.

Approximate budget requirements for analysis: Instrumentation needed for this method includes either a clinical analyser (approximately US$ 100 000) or a plate-washer, plate-reader, and various pipettes (approximately US$ 30 000). The cost for materials and supplies is around US$ 2–5 per sample for a commercial kit assay. Material costs may be slightly lower for laboratory-developed ELISA assays that measure serum ferritin in addition to other micronutrients.

Interpretation of results: The classification of iron stores based on serum ferritin concentrations by the presence or absence of inflammation are presented in Table 3.5. The generally accepted serum ferritin cutoff value for defining depleted iron stores is <15 μg/L for children over 5 years of age, adolescents, adults and women in the first trimester of pregnancy, while a cutoff value of <12 μg/L is used for children under 5 years of age (60). These cutoffs are appropriate when a regression or arithmetic correction approach has been applied to the ferritin concentrations to account for inflammation. If no mathematical correction for inflammation is applied to the ferritin concentrations, then the higher concentrations may be used to define deficiency (e.g., <30 μg/L or <70 μg/L).

Thresholds for elevated serum ferritin to identify risk of iron overload should be used only in clinical care with additional indicators and evaluation to establish the underlying cause.

Table 3.5. Recommended cutoffs to define iron deficiency in apparently healthy and non-healthy individuals by age group ^a

	Serum ferritin (μg/L)b
	Apparently healthy individualsc	Individuals with infection or inflammation
Infants and young children 0-4 years of age	<12	<30
Children and adolescents 5-19 years of age	<15	<70
Adults ≥20 years	<15	<70
Pregnant women first trimesterd	<15	<70

^a Source: reference ³.

^b Markers of inflammation should be assessed along with the ferritin concentration, and ferritin adjusted as necessary.

^c For the purposes of this guideline, an apparently healthy individual is defined as an individual with physical well-being for their age and physiological status, without detectable diseases or infirmities.

^d There are several physiological changes occurring in pregnancy that may contribute to the variation in thresholds of iron deficiency in pregnancy as defined by serum ferritin, including a physiological rise in acute phase proteins secondary to pregnancy; second trimester plasma volume expansion; and changes in inflammatory measures in the final trimester of pregnancy.

Transferrin receptor

Transferrin receptor is found on the cell membrane and allows iron-bound transferrin to enter the cell. When the iron supply is inadequate, the number of transferrin receptors on a cell surface increases. This enables the cell to compete more effectively for iron. The number of membrane receptors is in proportion to the soluble transferrin receptor (sTfR), a truncated form of transferrin receptor found in plasma. An increase in sTfR levels is seen in patients with iron deficient erythropoiesis or iron deficiency anaemia with increased erythropoiesis ¹⁴.

Because sTfR is not an acute phase protein, it may be less influenced by inflammation than ferritin. Other advantages of sTfR are that cutoff values do not vary with age or gender, or by physiologic state. However, the BRINDA project found that the relationship between sTfR and AGP was consistently significant and therefore recommended adjusting sTfR for AGP and malaria ¹⁵. The BRINDA project reported that the relationship between sTfR and CRP was most often not statistically significant. The decision to include malaria in the BRINDA adjustment was based on the physiologic response of sTfR during infection. Circulating sTfR levels may be elevated when there is increased red blood cell production or turnover or both, such as in the case of haemolytic anaemia ¹⁴.

Specimen collection and management: Most commonly, sTfR is measured in serum samples that are obtained by centrifugation from whole blood collected by venipuncture or capillary sample. Whole blood should be refrigerated as soon as possible and processed within a few days of blood collection. The serum is stable for at least one week at 4˚C, and for at least one year at -20˚C ⁶.

Biomarker analysis: sTfR is measured by immunoassays (clinical analyser or ELISA assay), most often using commercial assay kits. The required analysis volume is typically <25 µL serum, however a minimum specimen volume of >150 µL serum may be needed to fill the sample cup for the clinical analyser. The product sheet for the intended assay will specify the specimen matrix requirements and should be consulted before deciding on the method and ordering survey supplies. Serum is the preferred matrix, since not all assays can utilize EDTA or heparin plasma. Sandwich ELISAs are also available to measure sTfR along with other indicators, including those assessing other iron indicators, vitamin A and inflammation status ⁸. The method imprecision is usually around 10%.

A WHO-developed serum-based reference reagent (recombinant sTfR RR 07/202) is available through the NIBSC. This reagent has an assigned value based on protein content because assays have not yet been standardized and assay results are not comparable. The US College of American Pathologists proficiency testing offers a performance programme for laboratories performing sTfR measurements. CDC’s Performance Verification Program for Serum Micronutrients ¹⁰ covers sTfR and CDC also offers quality control materials for serum sTfR to support in-house quality assurance programmes for laboratories engaged in public health work ¹².

Approximate budget requirements for analysis: sTfR is measured on the same instrumentation as serum ferritin, however the cost for materials and supplies is higher (approximately US$ 5–10 per sample for a commercial kit assay). Material costs may be slightly lower for in-house developed ELISA assays that measure serum sTfR in addition to other micronutrients.

Interpretation of results: Assay-specific normal ranges for sTfR are available (for example, 2.9–8.3 mg/L for one brand of ELISA), however, there is no universally agreed normal range for sTfR. Similarly, there are no WHO definitions of public health problems based on sTfR prevalence. Using data generated with the Roche sTfR assay for the United States population in the National Health and Examination Survey (NHANES), cutoff values indicating iron deficiency were proposed as ≥6.0 mg/L for children 1-5 years and ≥5.3 mg/L for non-pregnant women 15-49 years ¹⁶.

Body iron index

The body iron index provides a quantitative assessment of body iron stores (index value >0 mg/kg) and indicates the size of the functional iron deficit. The functional deficit can be described as the amount of iron needed before it can be accumulated in the body’s stores, in an individual who is iron deficient (index value ≤0 mg/kg). The index is not a measure of total iron in the body. Previous terms used to describe this measure include “body iron”, “total body iron”, and “total body iron stores”.

In a controlled phlebotomy study, the sTfR only increased once the iron stores (measured by serum ferritin) were completely exhausted ¹⁷. When serum ferritin fell below 12 μg/L, the sTfR began to rise, roughly in proportion to the deficit in functional iron. This indicates that sTfR measures the deficit in tissue iron once stores are depleted. The combination of serum ferritin and sTfR levels can portray the spectrum of iron status from normal to severe deficiency. The formula to calculate the body iron index using ferritin and sTfR values (adjusted for measurement using the Ramco ELISA assay) in µg/L is as follows ¹⁸:

Body iron index (mg/kg) = –[log(Ramco sTfR/ferritin) – 2.8229]/0.1207

If the sTfR has been measured by the Roche assay instead of the Ramco ELISA assay, the relationship of that assay to the Ramco ELISA assay needs to be taken into consideration. The adjustment equation is as follows ¹⁹:

Ramco sTfR = 1.5 × Roche sTfR + 0.35 mg/L.

Folate

Folate is a generic term for several forms of folate with vitamin activity. Serum and red blood cell folate are key indicators used to assess folate status. Folate deficiency causes macrocytic anaemia, and low folate status among women increases the risk of bearing a child with neural tube defects. It is useful to consult the 2016 Biomarkers of Nutrition for Development (BOND) review for folate ¹ when considering indicators for folate assessment and analytic methods.

Because folates are sensitive to temperature, oxygen, and light, special consideration must be given to sample collection, processing, storage, and shipment.

Red blood cell (RBC) folate:

RBC folate indicates long-term folate status and is well correlated with liver folate stores. It is not affected by fasting and has traditionally been used to assess folate status in a population, both in terms of folate deficiency (risk of megaloblastic anaemia) and folate insufficiency (among women of reproductive age, risk for neural tube defects in the baby) ².

Specimen collection and management: Whole blood specimens need to be protected from light and from elevated temperatures to avoid folate losses ³ and must be stored in a refrigerator or a cold box with ice packs. It is best to process them on the day of collection, and they should be processed within 48 hours after collection.

A whole blood haemolysate needs to be prepared. For this, exactly 100 µL of carefully mixed EDTA whole blood (blood collection tube inverted 8–10 times) is added to a vial containing 1 mL of ascorbic acid solution (1% weight/volume). The vial with the haemolysate is mixed well and stored immediately in a -20°C freezer, for a maximum of one month, and shipped on dry ice to a laboratory for analysis. For storage beyond one month, the haemolysate must be stored at -70˚C.

If dried blood spots are generated in the field, the cards need to be completely dry prior to storing them in re-sealable plastic bags with desiccant sachets. Cards can be kept refrigerated for a maximum of one week; for a longer period, they need to be frozen at -20˚C or colder to avoid folate losses ⁴.

Biomarker analysis: RBC folate concentration can be measured using a variety of assays, including microbiologic assay, protein-binding assay, and high-performance liquid chromatography coupled to tandem mass spectrometry. There are also various methods within these assay types. WHO recommends the microbiologic assay because it requires the fewest resources to generate accurate results ². WHO thresholds for RBC folate are specific to the microbiologic assay and may not apply to other methods ^5,6,7. The microbiologic assay measures “total” folate and does not distinguish among various folate forms. Protein-binding assays also measure “total” folate, although several manufacturers have stopped marketing clinical RBC folate assays due to questions of assay validity and comparability, combined with decreasing demand. Chromatography-based assays differentiate individual folate forms, but it is a highly resource intensive approach that is not suited for laboratories with limited capacity and infrastructure.

The volume required for analysis is largely dependent on the assay used. While the microbiologic assay only needs about 25 µL per test, the other techniques mentioned generally need >100 µL per test. To allow for potential repeat analysis, the microbiologic assay also requires a minimum specimen of ≥100 µL.

Haematocrit or haemoglobin values and serum folate values (if available) can be used to calculate the RBC folate value using the following formula, where for example, a haematocrit of 40% would be entered into the equation as 0.4:

RBC folate = [whole blood folate – serum folate (1 – haematocrit)]/haematocrit

However, RBC folate values can be calculated if only the haematocrit or haemoglobin values are available by ignoring the serum folate contribution. This approach leads to slightly overestimated RBC folate values ⁸.

Dried blood spots can be used instead of whole blood haemolysates for the microbiologic assay, but generally not for the other mentioned techniques. Using the microbiologic assay, folate and haemoglobin are measured in the extract of the dried blood spot, and a haemoglobin-folate value is calculated (nmol folate per g haemoglobin). This value can be converted to RBC folate by multiplying haemoglobin-folate by the mean corpuscular haemoglobin concentration (MCHC, or g haemoglobin per L whole blood). An average MCHC value of 345 g/L can be used instead of individual MCHC values ⁴.

It is recommended to establish “in-house” whole blood haemolysate quality control materials that can be tracked over a longer period to verify that the method did not shift over time. The method imprecision for the microbiologic assay is around 10%, while for clinical analyser assays it is 5–10%. A lyophilized whole blood reference material is available from NIBSC (IS 95/528) with an assigned consensus value from multiple assays ⁹. Large assay differences that can exceed 100% can be observed in proficiency testing programmes, such as the CAP Ligand Survey ¹⁰ and the UK NEQAS programme ¹¹. CDC’s Performance Verification Program for Serum Micronutrients ¹² covers folate microbiologic assays and CDC also offers quality control materials to support in-house quality assurance programmes for laboratories engaged in public health work ¹³.

Approximate budget requirements for analysis: Instrumentation needed for the microbiologic assay includes a plate reader, an incubator, and various pipettes and small equipment (approximately US$ 40 000). Sample dilution and pipetting can be automated to increase sample throughput and reduce laboratory errors, however this requires additional resources (around US$ 50 000). The cost for materials and supplies is approximately US$ 2 per sample. The instrumentation cost for a clinical analyser can vary widely but is typically around US$ 100 000. The cost for commercial kits is approximately US$ 2–5 per sample.

Interpretation of results for RBC folate is described below in the section on serum folate.

Serum folate:

Serum folate represents recent folate intake. The serum folate status of an individual can only be interpreted when the specimen has been collected in a fasted state. However, population status can be interpreted from non-fasted samples because on average serum folate concentrations are only about 10% higher in non-fasted compared to fasted individuals ¹⁴.

Specimen collection and management: For serum folate, blood is collected in a blood collection tube without an anticoagulant. To avoid folate losses, the whole blood needs to be protected from light and from elevated temperature, thus stored in a refrigerator or in a cold box with ice packs. Storage of unprocessed whole blood at room temperature is unacceptable ^{3, 15}. It is best to process them on the day of collection, and they should be processed within 48 hours after collection.

Samples are centrifuged, and the serum is stored frozen until shipped on dry ice to a laboratory for analysis. Serum folate is stable for maximum one week at 4˚C and maximum one month at -20˚C, but for long-term storage the sample needs to be at -70˚C ¹². While serum is the preferred matrix, EDTA or heparin plasma can usually be used, however folate in EDTA plasma is particularly sensitive to oxidative losses at ambient or elevated temperatures. After the whole blood haemolysate is generated, the EDTA whole blood collection tube can be centrifuged to collect the plasma, if needed, which should be stored under the same conditions as specified for serum.

Biomarker analysis: The same assays used for RBC folate are employed to measure serum folate concentrations. Similar to RBC folate, WHO recommends the microbiologic assay for serum folate. Because serum folate concentrations are about 20 times lower than RBC folate concentrations, the sample dilution factor for serum is lower than for whole blood haemolysates. Serum and whole blood haemolysate specimens must be carefully diluted using calibrated pipettes to ensure accurate results ¹⁶.

A sufficient quantity of “In-house” serum quality control materials is needed to track assay performance over a longer time period than would be done with commercial materials. Serum-based reference materials are available from NIST (SRM 1955 and SRM 1950) and from NIBSC (IS 03/178) ⁹. However, none of these materials has certified values for total folate, which is what the microbiologic assay measures and is considered the gold standard method. Assay differences for serum folate are somewhat smaller than for RBC folate, but they can still be in the range of 30–50%, as can be observed in proficiency testing programmes such as the CAP Ligand Survey ¹⁰. CDC’s Performance Verification Program for Serum Micronutrients ¹² and quality control materials for folate support in-house quality assurance programmes for laboratories engaged in public health work ¹³.

Approximate budget requirements for analysis: The same resources and instrumentation described in the section on RBC folate are needed for the measurement of serum folate, and costs are similar.

Interpretation of results: The cutoff values for folate status in all age groups, using macrocytic anaemia as a haematological indicator, are presented in Table 3.7. Additional cutoff values for folate deficiency, based on rising homocysteine concentration as a metabolic indicator, of <14 nmol/L for serum folate and <624 nmol/L for RBC folate can be used with data produced from the microbiologic assay calibrated with 5-methyltetrahydrofolate, the main form of folate found in serum and RBCs. This data would serve to determine metabolic risk (elevated homocysteine) ¹⁷. Note that these values are not listed in the table. Table 3.8 shows the red blood cell folate cutoff values defined for the prevention of neural tube defect-affected pregnancies in women of reproductive age ⁷. These values are at the population level. WHO recommends the relevant mean population cutoff value as RBC folate <400 ng/mL or <906 nmol/L ⁷. The recommended value was derived from epidemiologic data produced using a microbiologic assay calibrated with folic acid. If ins#tead data are produced with a microbiologic assay calibrated with 5-methyltetrahydrofolate, a cutoff value of <748 nmol/L should be used ⁵. These thresholds should not be used at an individual level for determining risk of a neural tube defect-affected pregnancy, and there is no individual threshold to recommend ⁷.

Table 3.7. Folate concentrations in serum and red blood cells for determining individual-level folate status in all age groups, using macrocytic anaemia as the haematological indicator ^a

Serum/plasma folate levels ng/mL (nmol/L)b,c	Red blood cell folate level ng/mL (nmol/L) b,c	Interpretation
>20 (>45.3)		Elevated
6-20 (13.5-45.3)		Normal range
3-5.9 (6.8-13.4)		Possible deficiency
<3 (<6.8)	<100 (<226.5)	Deficiency

^a Source: reference ².

^bFolic acid conversion factor: 1 ng/mL = 2.265 nmol/L.

^c Assayed by Lactobacillus casei via microbiologic assay.

Table 3.8. Mean RBC folate concentrations in red blood cells for preventing neural tube defect-affected pregnancies in women of reproductive age at the population level ^a

Red blood cell (RBC) folate level, ng/mL (nmol/L) b,c	Interpretation
>400 (>906)	Folate sufficiencyt
≤ 400 (≤906)	Folate insufficiency

^a Source: reference ².

^bFolic acid conversion factor: 1 ng/mL = 2.265 nmol/L.

^c No individual level threshold is recommended for the prevention of neural tube defects in women of reproductive age.

Vitamin A

There are multiple indicators for determining vitamin A deficiency. The four most commonly used biological indicators are serum (or plasma) retinol, retinol-binding protein (RBP), and the modified relative dose response (MRDR). Breast milk retinol can be used in some circumstances.

WHO recommends the use of two different criteria for determining the presence and severity of vitamin A deficiency as a public health problem. One is when the population prevalence of at least two biological parameters from a range of functional and biochemical indicators, one of them being serum retinol, exceed the threshold for defining a public health problem ¹. The second criterion specifies one biological indicator with a prevalence below the population-level cutoff value and at least four ecologic risk factors for vitamin A deficiency, two of which should be nutrition- or diet-related ¹. Demographic or ecologic risk factors include nutrition-related and illness-related risk factors. Examples of relevant risk factors are:

Nutrition- and diet-related risk factors:

• <50% prevalence of breastfeeding in infants 6 months of age

• Median dietary vitamin A intake <50% of recommended safe levels of intake among 75% of children 1-6 years of age

• Stunting rate ≥30% and/or wasting rate ≥10% among children under 5 years of age

• Food frequency assessment findings that foods with high vitamin A content consumed <3 times per week by ≥75% of vulnerable groups

Illness-related risk factors:

• Infant mortality rate >75 per 1000 live births and child mortality rate of >100 per 1000 live births

• Full immunization coverage <50% of children between 12-23 months of age

• Two-week prevalence of diarrhoea of >20%

• Measles case fatality rate of ≥1%

• No formal schooling for >50% of women 15-44 years of age

• <50% of households with a safe water source (boiled, treated, filtered, properly stored)

Serum (or plasma) retinol:

WHO recommends that serum retinol be used along with either another biological indicator of vitamin A status or with the other risk factors listed above to define the degree of public health significance of vitamin A deficiency at the population (not individual) level and to assess the need for vitamin A interventions ^{1, 2}. Serum (or plasma) retinol can indicate subclinical, or marginal, vitamin A deficiency. Concentrations can change in response to vitamin A interventions when liver stores are low; when liver stores are replete, retinol concentrations may not respond to vitamin A interventions². Serum retinol also correlates with the prevalence and severity of xerophthalmia.

Serum retinol concentrations are homeostatically controlled, but inflammation does cause them to decrease. Without an accompanying indicator of inflammation, artificially depressed serum retinol concentrations may lead to an overestimation of vitamin A deficiency prevalence. Inclusion of indicators such as CRP or AGP are recommended in any survey that includes the assessment of serum retinol to identify individuals with inflammation. However, at the time of this writing, the BOND review states that there is no consensus on the need for, or best method to, adjust for identified inflammation ³.

Specimen collection and management: Most commonly, retinol is measured in serum samples that are obtained by centrifugation of whole blood collected by venipuncture or finger prick. Whole blood needs to be refrigerated immediately and centrifuged within a few days of collection. When protected from light, the vitamin A in serum is stable or at least one week at 4˚C and for at least one year at 20˚C ⁴. However, procedures such as centrifugation and an adequate cold chain can be difficult to implement in remote field conditions.

Biomarker analysis: The most common and accurate method for measuring serum retinol is high-performance liquid chromatography (HPLC) with UV detection. In an adapted “micromethod,” ⁵ the required analysis volume is only 25 µL of serum or plasma. The minimum specimen volume is 100 µL to provide enough sample for a repeat analysis if needed. EDTA or heparinized plasma can be used, but serum is the preferred matrix. Commercially available retinol with greater than 95% purity is used as a calibrator (HPLC-grade reagents are preferred). Retinyl acetate, which is also commercially available, is used as an internal standard to correct for variations during the analytical procedure.

Quality control (QC) measures: Analytical method imprecision is typically around 5%. Moderate assay differences can occur with analyses conducted in different laboratories, therefore laboratories should participate in an external quality assurance programme such as CDC’s Vitamin A Laboratory and External Quality Assurance (VITAL-EQA) programme ⁶, CDC’s Performance Verification Program for Serum Micronutrients ⁷, which provides a one-time or annual performance report, or the National Institute of Standards and Technology (NIST) Health Assessment Measurements Quality Assurance Programme (HAMQAP). Serum-based certified reference materials (multiple levels of Standard Reference Material® (SRM®) 968) are available from NIST (Gaithersburg, MD, USA) to verify method accuracy. Quality control materials for serum micronutrients including retinol are available from CDC to support in-house quality assurance programmes for laboratories engaged in public health work ⁸.

Approximate budget requirements for analysis: Instrumentation needed for this method includes an HPLC and UV detector, centrifuge, vortex, and various pipettes (costing approximately US$ 50 000). The cost for materials and supplies is approximately US$ 5 per sample.

Interpretation of results: Low serum retinol is defined as <0.70 µmol/L in children 6–71 months of age. A serum retinol cutoff value of <1.05 mol/L is sometimes used to indicate vitamin A insufficiency. Interpretation of a population’s prevalence of serum retinol concentrations <0.70 µmol/L for defining a public health problem is presented in Table 3.1. It is important to note that this is based on low serum retinol values that have not been adjusted for inflammation.

Table 3.1. Prevalence of low serum retinol (<0.70 μmol/L),^a used in conjunction with another indicator,^b to define a public health problem at the population level and its level of importance among children 6–71 months of age ²

Level of importance as a public health problem	Prevalence
Mild	2 to 9%
Moderate	10 to 19%
Severe	≥20%

^aEquivalent to serum retinol <20 μg/dL. Prevalence estimates based on retinol values that have not been adjusted for inflammation

^b“Another indictor” pertains to either a second biomarker for vitamin A deficiency, or at least four ecologic indicators for vitamin A deficiency, two of which should be nutrition or diet related.

Retinol Binding Protein (RBP):

RBP can be used as a surrogate, or proxy, indicator for retinol ³; however, a 1:1 molar equivalence between retinol and RBP does not usually occur. This means that the serum retinol cutoff value cannot be applied to RBP. The currently recommended approach to calculating an RBP cutoff value, detailed in the BOND review ³ and practiced in survey reports ^9,10, is to measure serum retinol concentrations by HPLC in a subsample of the population where RBP is being used to assess vitamin A deficiency. Within the retinol subsample, given a reasonable correlation between RBP and retinol, a linear regression model is used to calculate an RBP concentration equivalent to retinol of 0.7 µmol/L. Ideally, the same blood draw would be used for measuring retinol and RBP (as opposed to taking a morning collection for RBP and an afternoon collection from the same person for retinol, for example). Outliers should be removed when reviewing the serum retinol-RBP regression plot, using studentized residuals larger than 3 in absolute value ¹¹. A minimum sample size for the retinol subsample is suggested to be 20% of the total survey population or at least 100 observations, selected at random. The field of vitamin A assessment is rapidly evolving; the guidance presented in this manual is based on the best evidence available at the time of publication.

Specimen collection and management: RBP can be measured in serum or plasma. Blood should be collected, processed and stored as noted for the collection and management of specimens for serum (or plasma) retinol. The BOND Expert Panel for Vitamin A assigned the same degree of difficulty to serum RBP and serum retinol for sample collection and sample transportation. The dried blood spot (DBS) methodology for measuring serum retinol and RBP is less reliable than the method based on serum or plasma ¹².

Biomarker analysis: Several commercial ELISA methods and laboratory-developed tests are available to measure RBP in serum or plasma, with serum being the preferred specimen matrix. However, no certified reference materials are available to verify RBP method accuracy, and RBP is therefore not included in external quality assurance programmes. Notably, the CDC VITAL-EQA programme ⁶ includes quality assurance for retinol, as well as for other nutritional biomarkers. In this programme, RBP is compared to retinol. Similar to using RBP as a proxy for retinol in surveys, laboratories that participate in VITAL-EQA may also compare RBP to retinol. Thus, although there are no external quality assurance programmes for RBP there is the opportunity to measure RBP in serum or plasma in commercial ELISA methods and compare to laboratory-developed tests against retinol as a proxy. Another advantage of these results is sample comparability because both biomarkers will be performed on the same specimen, handled under the same conditions. The specimen should be gently and thoroughly mixed before measuring the retinol and RBP.

Approximate budget requirements for analysis: Instrumentation needed for ELISA methods includes a plate washer, plate reader and various pipettes (approximately US$ 30 000). The cost for materials and supplies is approximately US$ 2–5 per sample, depending on whether the assay used is laboratory-developed or a commercial kit. For a laboratory developed RBP test, a significant amount of time will be necessary to find appropriate antibodies and validate the assay.

Interpretation of results: There are at present no WHO guidelines on the interpretation of vitamin A deficiency prevalence based solely on RBP. When RBP is measured in surveys as a proxy for retinol, it is important to include a caveat if the public health significance of vitamin A deficiency is based on a prevalence of RBP <0.7 µmol/L. Another important consideration to keep in mind is that the prevalence numbers in Table 3.1 are based on serum retinol values that have not been adjusted for inflammation. It is recommended to present both inflammation-adjusted and unadjusted estimates for vitamin A deficiency until guidance from WHO becomes available. Suggested methods for adjusting serum retinol concentrations based on CRP and AGP include the use of regression or arithmetic correction factors, such as those developed by BRINDA ¹³ and Thurnham ¹⁴, respectively. Also, since enzyme immunoassays do not distinguish between holo- and apo-RBP, an additional adjustment (that requires determination of serum retinol) is needed to reflect the RBP:retinol ratio in the population of interest ^{15, 16}. Box 3.2 summarizes the use of RBP for assessing vitamin A status.

Box 3.2 Summary of the use of RBP

At the time of this writing, the preferred method for assessing vitamin A status is serum retinol. When RBP is selected as the main vitamin A indicator for a survey, it is also necessary to measure retinol in a subsample of the population. This will permit the determination of a survey specific RBP cutoff value to define vitamin A deficiency. It is also useful to measure MRDR in a subsample because it provides an assessment of vitamin A liver reserves and the WHO recommendation of two biomarkers to assess deficiency.

It may be complicated to analyse trends if there are different survey-specific population-level cutoff values within the same country from various years. For example, the RBP survey-specific cutoff value to define vitamin A deficiency may be 0.78 µmol/L in one survey cycle and 0.58 µmol/L in the next survey cycle. As such, the prevalence of RBP below those two separate cutoff values may be hard to compare and raises the question of why the RBP-retinol relationship changed in a population between survey cycles. This is important to note, and may be a factor to consider when choosing vitamin A indicators. WHO has defined the retinol cutoff value for deficiency, making it easier to assess trends over time when retinol is collected and analysed using similar methods across surveys. However, many older surveys did not assess indicators of inflammation, which can influence the interpretation of retinol and trends over time as the prevalence of inflammation can vary from survey to survey.

Modified relative dose response (MRDR):

RBP is synthesized in the liver as apo-RBP (unbound RBP). When liver reserves of vitamin A are low, apo-RBP accumulates in the liver. When vitamin A becomes available from newly ingested sources the accumulated apo-RBP binds to the retinol and is released into circulation as the holo-RBP complex (retinol bound to RBP). MRDR is a functional test that takes advantage of this process by providing individuals with a measurable “challenge” dose of 3,4-didehydroretinol (also known as DR or vitamin A2) in the acetate form. DR binds to apo-RBP in the liver forming the holo-RBP complex, which is quickly released into the plasma during deficiency. After this challenge dose, DR should appear in serum in significant amounts over baseline (prior to the challenge dose) only when liver reserves of vitamin A are low. Therefore, the amount of DR released is an indication of vitamin A status. MRDR is calculated from the molar ratio of DR to retinol (DR:R). In comparison with other vitamin A indicators, MRDR is less influenced by inflammation and it is not homeostatically controlled in the timeframe of the test ³. An important consideration of MRDR is the time required between administering the challenge dose and collecting the specimen (4-6 hours after the challenge).

The MRDR is useful to assess changes in liver stores of vitamin A, for example, changes in response to an intervention to improve vitamin A status. The MRDR test provides useful semi-quantitative information to evaluate deficiency through low liver reserves of vitamin A. On the contrary, it is not useful in defining excessive vitamin A reserves. MRDR is recommended for inclusion among a randomly selected subsample of individuals, to assess the underlying vitamin A status of the population studied ³. Serum retinol is collected and analysed from the same blood draw as that used for MRDR. When assessing vitamin A deficiency at the population level, it is useful to assess the mean and standard deviation of the MRDR value (namely, DR:R). When comparing results from MRDR with results from RBP and retinol, there may be inconsistencies at the individual level for categorizing deficiency. Thus, comparing vitamin A deficiency prevalence estimates from MRDR, RBP, and retinol may cause confusion. It is most useful to look at two biologic parameters (MRDR plus either retinol (preferred) or RBP) to determine the population status of vitamin A deficiency as a public health problem ^{3, 1}.

Specimen collection and management: In preparation for specimens for an MRDR assessment, an individual must consume a small challenge dose of a retinol analog (DR or vitamin A2) along with a fatty snack (lacking in vitamin A) to ensure absorption. This should be done about 4 to 6 hours before collecting 1-3 mL of venous blood. The dose of vitamin A2 can be mixed with 1 mL of olive oil or another edible oil containing no vitamin A. Administering it using a disposable syringe helps ensure that it is completely swallowed, especially with small children. Survey participants must also be questioned about recent consumption of foods rich in vitamin A prior to administering the vitamin A challenge dose. If there has been recent consumption of vitamin A rich foods, it will be necessary to wait two hours before proceeding with the test. Vitamin A rich foods should not be consumed again until after the blood draw for the MRDR test.

The same procedures for transporting, processing, and storing of specimens for other vitamin A indicators apply to the MRDR venous blood specimen.

Biomarker analysis: Analysis of 3,4-di-dehydroretinol requires HPLC and can be assessed in the same analytical run as serum retinol. The required sample volume for analysing serum retinol and 3,4-di-dehydroretinol is 250 µL of serum or plasma, and the minimum specimen volume is 500 µL to provide enough sample for a repeat analysis if needed. Retinol acetate is used as an internal standard calibrator for retinol (commercially available at a satisfactory purity >95%) in each analytical run. Quality control (QC) samples are recommended, which have a known concentration of retinol that covers the range of retinol concentrations expected in the human population (low, medium and high) and are used to validate each analytical assessment, aiding in correcting analytical bias.

Approximate budget requirements for analysis: Instrumentation needed for this method includes an HPLC, centrifuge, vortex, and various pipettes (costing approximately US$ 50 000 for the complete set of equipment). The cost for materials and supplies is approximately US$ 5 per sample.

Interpretation of results: MRDR is a semi-quantitative indicator of vitamin A status. The MRDR value, which is the ratio of DR to retinol in serum, indicates adequacy of liver reserves. For individuals, the 2016 BOND review ³ recommends a MRDR cutoff value of ≥0.060 to indicate insufficient liver reserves (≤0.1 µmol retinol/g liver vitamin A), and of <0.060 to indicate enough liver reserves (≥0.1 µmol retinol/g liver vitamin A). For groups, a mean MRDR value <0.030 is recommended for indicating adequate vitamin A status ³.

Breast milk retinol:

WHO recommends exclusive breastfeeding for infants in the first 6 months of life, followed by continued breastfeeding with appropriate complementary foods for up to 2 years or beyond ¹⁷. Breast milk retinol concentrations provide information about both the mother and breastfed infant. They are considered to reflect the recent dietary intake of mothers and can be used to estimate vitamin A intake of infants receiving the breast milk ^{3, 18}. Breast milk retinol concentrations have been used to assess the risk of vitamin A deficiency in populations, determine the efficacy of maternal vitamin A interventions, and for the monitoring and evaluation of programmes providing maternal vitamin A interventions ³. Average breast milk retinol concentrations from well-nourished women are about 485 µg/L ¹⁹; however, average concentrations can fall below 400 µg/L in areas where vitamin A deficiency is of public health significance ¹. When selecting the population for evaluation, age, stage of lactation, geographic location, season and pregnancy status should be considered ³. Vitamin A content is very high in colostrum (milk secreted in the first 4-6 days postpartum), and remains high in transitional milk (days 7-21 postpartum), after stabilizing in mature milk (after about day 21 postpartum). Therefore, breast milk samples collected after one month postpartum, avoiding colostrum and transitional milk samples, are most useful for assessment of vitamin A status.

Specimen collection and management: Breast milk can be collected as a full milk sample or as a casual sample. The specimen collection method would depend on the survey objectives. A casual sample is appropriate for assessment of population-level prevalence of low breast milk vitamin A, expressed in nmol retinol/g fat, and will be described here. A survey objective of estimating the vitamin A intake of infants from breast milk would necessitate that a full milk sample be collected, which is described in detail elsewhere ²⁰.

Milk collected using the casual sample method (~10 mL) can be hand-expressed into specimen cups or tubes made of polypropylene. A benefit of casual milk collection is that there is no need to standardize ‘time since last feed’; however, milk fat will need to be measured. One option for milk fat measurement is a creamatocrit centrifuge, which is field friendly. Casual milk collection is defined as mid-feed collection 1 minute after let-down, by manual expression. Although women can do the manual expression in privacy and without assistance after receiving adequate instructions, it is important to consider the gender of the field staff and the local context as female health workers may be more appropriate for surveys that include breast milk collection. The breast milk needs to be refrigerated at 4˚C immediately and protected from direct light because vitamin A in milk is less stable than serum retinol, which is protein bound ²⁰. Protection from light and keeping the milk cold will prevent photodegradation of the vitamin A. If refrigerated, the breast milk must be analysed within 24 hours of collection or it may be stored frozen at 20˚C (or colder, such as 80˚C) and analysed within one year of collection ³. Before freezing, precise aliquots of milk that will be used for measuring vitamin A content should be prepared as thawed samples can be difficult to homogenize. However, procedures such as centrifugation and an adequate cold chain can be difficult to implement in remote field conditions.

Biomarker analysis: The most common and accurate method for measuring breast milk retinol is high-performance liquid chromatography (HPLC) with UV detection after saponification ²¹. Portable fluorometers are also field friendly equipment that enables mothers to get immediate results on their breast milk vitamin A status ²², and has performed well compared to HPLC ²³. Because vitamin A is found in the milk fat, fresh milk should be mixed well so that the cream layer is evenly distributed within the sample taken for measurement. A sample volume of 2 mL of breast milk is required for analysis using HPLC. The minimum specimen volume is 100 µL to provide enough sample for a repeat analysis if needed. Commercially available retinol with greater than 95% purity is used as a calibrator (HPLC-grade reagents are preferred). The base solution to be used for saponification should be mixed and stored in plastic containers to remain stable ²¹. Either 3,4-didehydroretinyl acetate (3), C23-beta-apo-carotenol ^{24, 25}, or tocol ²⁶, may be used as an internal standard to correct for variations during the analytical procedure. To determine the amount of milk fat in a specimen, the creamatocrit methods can be done in a laboratory ²⁷, or a creamatocrit centrifuge can be used in the field.

Quality control (QC) measures: Analytical method imprecision is typically around 5%. Moderate assay differences can occur with analyses conducted across laboratories; however, the National Institute of Standards and Technology (NIST) Health Assessment Measurements Quality Assurance Programme (HAMQAP) does not have certified control for human breast milk, making external quality assurance impractical.

Approximate budget requirements for analysis: Instrumentation needed for the HPLC method includes an HPLC with a UV detector, centrifuge, vortex, and various pipettes (costing approximately US$ 50 000). A calibrated spectrophotometer is also needed for external standard quantification. The cost for materials and supplies is approximately US$ 5 per sample.

Interpretation of results: Breast milk retinol concentrations ≤1.05 µmol/L are considered inadequate (18), but this cutoff applies only to full milk collection. It is preferable to express breast milk retinol concentrations per gram of fat to account for fat variability. Retinol concentrations ≤28 nmol/g milk fat (or ≤8 µg/g milk fat) are considered inadequate (18). Vitamin A deficiency is considered a public health problem of mild, moderate or severe importance at a prevalence of inadequate concentrations of <10%, 10-24%, and ≥25%, respectively.

Clinical and functional indicators:

Clinical or functional indicators of vitamin A deficiency usually focus on xerophthalmia, an eye condition that worsens as the depletion of vitamin A stores progresses ²⁸. Most of these indicators are not recommended for routine cross-sectional surveys due to their rare occurrence, even in areas endemic for vitamin A deficiency. The one exception is assessing whether women have experienced night blindness during a pregnancy within the previous three years or five years. To help interpret reported vision problems, women being assessed should report problems seeing at night as well as during the day during the last pregnancy. The WHO/International Vitamin A Consultative Group (IVACG) states that a prevalence of night blindness that exceeds 5% among pregnant women would indicate vitamin A deficiency of public health significance among the population ²⁹.

Micronutrient and related health indicators

This section describes indicators for selected micronutrients (iron, iodine, folate and vitamins A, B12, and D) and related health issues (anaemia, iron deficiency and iron deficiency anaemia).

Throughout the section, recommended cutoff values for defining deficiency or insufficiency are listed. It is important to note that cutoffs may not be available for all population groups of interest. In addition, inflammation is an area of current research that affects micronutrient measures and new methods to adjust for inflammation are being explored.

Cutoff values for populations groups

Given the higher nutrient demands required for growth and reproduction, young children and women of reproductive age are the most vulnerable population groups and are thus the most common targets of nutrition surveys.

For pregnant women, unique cutoff values are only available for haemoglobin (anaemia), ferritin during the first trimester (iron deficiency) and urinary iodine. No cutoff values specific to this group are available for other micronutrients, and research is not sufficient to determine whether such values are needed. If stable representative estimates for pregnant women are needed in a survey, then this group will need to be oversampled and the results categorized separately.

Box 3.1 BOND papers available as of June 2021

Vitamin B12
Allen LH, Miller JW, de Groot L, Rosenberg IH, Smith AD, Refsum H et al. Biomarkers of Nutrition for Development (BOND): vitamin B-12 review. J Nutr. 2018;148(suppl_4):1995S-2027S. doi: 10.1093/jn/nxy201 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6297555/) ¹.

Iron
Lynch S, Pfeiffer CM, Georgieff MK, Brittenham G, Fairweather-Tait S, Hurrell RF et al. Biomarkers of Nutrition for Development (BOND) – iron review. J Nutr. 2018;148(suppl_1):1001S-1067S. doi: 10.1093/jn/nxx036 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6297556/) ².

Vitamin A
Tanumihardjo SA, Russell RM, Stephensen CB, Gannon BM, Craft NE, Haskell MJ et al. Biomarkers of Nutrition for Development (BOND) – vitamin A review. J Nutr. 2016;146:1816S-48S. doi: 10.3945/jn.115.229708 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4997277/) ³.

Zinc
King JC, Brown KH, Gibson RS, Krebs NF, Lowe NM, Siekmann JH et al. Biomarkers of Nutrition for Development (BOND) - zinc review. J Nutr. 2016;146:858S-885S (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4807640/) ⁴.

Folate
Bailey LB, Stover PJ, McNulty H, Fenech MF, Gregory JF 3rd, Mills JL et al. Biomarkers of Nutrition for Development -folate review. J Nutr. 2015;145:1636S-1680S. doi: 10.3945/jn.114.206599 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4478945/) ⁵.

Iodine
Rohner F, Zimmermann M, Jooste P, Pandav C, Caldwell K, Raghavan R, Raiten DJ. Biomarkers of Nutrition for Development – iodine review. J Nutr. 2014;144:1322S-1342S. doi: 10.3945/jn.113.181974 (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4093988/) ⁶.

These papers are also included in the reference list at the end of this module.

Inflammation

The acute phase response, triggered by infection and trauma, is a collection of non-specific changes including the production of proteins that promote inflammation and activate, complement, and stimulate phagocytic cells. All of these are inflammatory markers. This cascade of immune response activity is intended to remove harmful molecules and pathogens and to prevent further damage to tissues ⁷.

Inflammation affects the circulating concentrations of multiple micronutrients. During the acute phase response there is a change in many indicators of micronutrient status, such as retinol and ferritin, leading to an over- or under-estimation of deficiency. The acute phase proteins, C-reactive protein (CRP) and alpha-1-acid glycoprotein (AGP), are commonly used indicators of inflammation ⁷. The concentration of some acute phase proteins (APPs) in plasma, called positive APPs, will increase in the presence of inflammation. Examples of such APPs include CRP, AGP, and ferritin. Other APPs decrease in the presence of inflammation. These negative APPs include retinol, retinol binding protein (RBP), and albumin.

The BRINDA project (Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia) has been investigating approaches to adjust population estimates of iron, vitamin A, and zinc in the presence of inflammation. The progress for iron and vitamin A has been published, and work on zinc is ongoing. In general, BRINDA described methods that incorporate an internal (country- or survey-specific) regression-correction approach ⁸. This is a rapidly evolving area and it is important to review the literature for updated information. The BRINDA website also contains useful resources ⁹.

In many lower income countries, the prevalence of infection and resultant inflammation is high, and many individuals will have elevated APPs even without clinical signs or symptoms of disease. Additionally, chronic conditions without infection, such as diabetes, hypertension and obesity, result in low grade inflammation.

Measuring inflammation by CRP:

CRP concentration increases rapidly during inflammatory processes, and returns to pre-infection levels over 18-20 hours when the stimuli end, which is more quickly than AGP ⁷.

Specimen collection and management: CRP is typically measured in serum samples obtained by centrifuging whole blood that was collected by venipuncture or capillary sampling. CRP is stable, so whole blood processing can be delayed for 1-2 days. Serum is stable for up to two weeks at 4˚C and up to one year at -20˚C.

Biomarker analysis: CRP and high-sensitivity CRP (hs-CRP) ^{10, 11} are measured by immunoassays (nephelometry or turbidimetry), either on a fully automated clinical analyser or by using a manual ELISA assay. Commercial assay kits are available for both analytical techniques. The required analysis volume is normally <25 µL; however, a minimum specimen volume of >150 µL may be needed to fill the sample cup for the clinical analyser. The assay product sheet will explain matrix requirements. Not all assays can utilize EDTA (ethylenediaminetetraacetic acid) or heparin plasma. Serum is the preferred matrix.

Quality control: Appropriate quality control measures must be followed to ensure high quality results. The assay kits include calibration materials and may include quality control materials. It is nonetheless recommended to establish “in-house” quality control materials that can be tracked over a longer period to verify that the method did not shift over time. The method imprecision is typically ~10%. A human serum international reference material (ERM-DA474/IFCC) is available through the Institute for Reference Materials and Measurements (IRMM) at the European Commission Joint Research Centre. However, not every assay may be able to use this material because the assay performance may differ between patient samples and reference materials that have undergone some processing. Moderate differences between assays can be observed in proficiency testing programmes, such as the Immunology Survey of the US College of American Pathologists (CAP) and the National Institute of Standards and Technology (NIST).

Approximate budget requirements for analysis: Approximate budget requirements for analysis: Instrumentation needed for CRP includes either a clinical analyser (approximately US$ 100 000) or a plate-washer, plate-reader, and various pipettes (approximately US$ 30 000). The cost for materials and supplies is roughly US$ 3 to US$ 5 per sample for a commercial kit assay. Material costs may be slightly lower for locally developed ELISA assays that measure CRP in addition to other micronutrients.

Interpretation of results: The suggested CRP cutoff value to define inflammation is >5 mg/L ⁷¹².

Measuring inflammation by AGP:

The concentration of AGP normally increases within 24 to 48 hours following an infection and stays elevated for a longer duration than CRP. Typically, it is elevated for 5 days, from day 2 to day 7 after a single clinical infection ^13,14,15. Hence, AGP captures a unique phase of the acute phase response, compared to CRP ⁷.

Specimen collection and management: Like CRP, AGP is stable, and specimens can be handled using similar conditions.

Biomarker analysis: The analytical techniques to measure AGP are the same as for CRP; however, there are fewer clinical analysers available that measure AGP. A human serum international reference material (ERM-DA470K/IFCC) is available from the European IRMM. AGP is usually not covered in proficiency testing programmes and it is therefore difficult to compare among assays.

Approximate budget requirements for analysis: The same resources and instrumentation described for CRP are needed for the measurement of AGP.

Interpretation of results: The suggested AGP cutoff value to define inflammation is >1 g/L ^7-12.

Note regarding stability of biomarkers in serum samples
The following sub-section refers to stability of the biomarker at 4oC and -20oC (16, 17). The section discusses the collection of specimens and management of serum samples for analysis of: retinol, retinol binding protein, ferritin, transferrin receptor, vitamin B12, 25-hydroxy vitamin D and CRP.

Anemia

Anaemia is usually defined by a low haemoglobin concentration adjusted for elevation, and (among adults) adjusted for smoking. One of the most common causes of anaemia is iron deficiency. However, anaemia cannot necessarily be used as a proxy for iron deficiency because anaemia can result from many other factors, including:

• Malaria and other infections
• Other causes of blood loss (such as heavy menses, haemorrhage in childbirth, trauma, gastrointestinal bleeding due to ulcers)
• Deficits in other nutrients (for example vitamin A, folic acid, vitamin B12)
• Haemoglobinopathies (such as sickle cell or thalassemia)
• Overweight, obesity and other causes of chronic inflammation (for example chronic kidney disease) ¹
• Blood loss due to infection (from such conditions as hookworm, schistosomiasis or H. pylori) ^{2, 3}.

Haemoglobin:

Haemoglobin level is an indicator of anaemia, a condition in which the number of red blood cells or their oxygen-carrying capacity is insufficient to meet physiologic needs. Iron deficiency is one of the most common causes of anaemia globally, although anaemia can also be caused by other conditions ⁴.

Additional haematologic indicators of anaemia include haematocrit, mean cell volume and red blood cell distribution width. It is recommended that haemoglobin be used for assessing anaemia in cross-sectional surveys. For assessing the prevalence of iron deficiency anaemia, it is recommended that countries collect data on haemoglobin and at least one biochemical test for iron deficiency, along with measures of inflammation as appropriate ⁵.

Specimen collection and management: Haemoglobin is typically measured in fresh whole blood samples in the field ⁶. For non-field-based analysis, haemoglobin is most commonly measured in EDTA blood samples. In this case, the samples should be refrigerated as soon as possible and need to be analysed within 1–2 days of collection.

In the case of field analysis, capillary drops, pooled capillary blood into small blood collection tubes, or venous blood can be used. Capillary blood from a finger prick is collected by capillary action in a cuvette, which is then placed in the photometer that displays the haemoglobin concentration within one minute. The accuracy of haemoglobin measurement may be improved by pooled capillary or venous blood. For pooled capillary blood, collect 250–500 μL in a small blood collection tube containing an anticoagulant such as EDTA or heparin, gently mixing the blood by inverting the tube several times to prevent clotting, and then filling the cuvette with blood from the blood collection tube ^{7, 8}. For venous blood, collect 3-5 mL in a vacuum blood collection tube containing an anticoagulant such as EDTA or heparin, gently mixing the blood by inverting the tube several times, and then filling the cuvette with blood from the collection tube for analysis ^{6, 8}. A comparison of nationally representative surveys measuring haemoglobin using HemoCue® with capillary (DHS) or venous (BRINDA) samples, showed substantial differences in anaemia prevalence estimates, which were consistently lower in venous compared to capillary ⁹.

The procedures for specimen collection and analysis using a haemoglobinometer must be standardized. This requires careful training of survey technicians. It is particularly important not to squeeze the finger too hard when collecting capillary blood because this can cause interstitial fluid to mix with the blood and result in an incorrect haemoglobin concentration. Poor quality collection of capillary blood can in turn lead to low or high haemoglobin concentrations for population-based surveys ⁶.

Biomarker analysis: The most commonly used method for field-based measurement of haemoglobin in population surveys is photometric determination using a portable haemoglobinometer ^{6, 9}. The procedure does not require specialized laboratory personnel and the haemoglobinometer may be operated on four AA batteries, which makes it particularly useful in the field. Manuals and tutorial videos for haemoglobinometers are available online, making it easier to follow proper operation ^{7, 8}. The HemoCue® haemoglobinometer has been validated against traditional haemoglobin laboratory methods and found to have adequate accuracy and precision in controlled settings ^{7, 8}. The accepted reference method for haemoglobin measurement is the cyanmethaemoglobin method ¹⁰.

A systematic review commissioned by WHO for reviewing haemoglobin cutoffs as part of the project to review cutoffs to diagnose anaemia concluded that capillary fingerprick blood usually produces higher haemoglobin concentrations compared with venous blood, that individual drops produced lower concentrations than pooled capillary blood and that compared to automated haematology analysers, other methods (cyanmethaemoglobin, WHO Colour Scale, paper‐based devices, HemoCue® Hb‐201 and Hb‐301, and Masimo Pronto®) overestimated haemoglobin concentrations ⁶.

Approximate budget requirements for analysis: Each haemoglobinometer costs approximately US$ 300–500 depending on the model, and the cuvette cost is approximately US$ 0.50 when both items are procured through UNICEF. While most of the field experience in the use of portable haemoglobinometers has been with HemoCue®, other portable haemoglobinometers are available from other manufacturers at a similar cost (US$ 400–600).

Adjustments and interpretation of results: WHO has established cutoff values for haemoglobin to define anaemia by population group, including for pregnant women ^{5, 11}. These are shown in Tables 3.2–3.4. Table 3.2 presents haemoglobin levels used to diagnose anaemia, while Table 3.3 defines the public health significance of anaemia in a population. Table 3.4 shows the adjustments of haemoglobin values that are required to correct for changes that occur due to elevation and smoking (based on the average number of cigarettes per day). Populations living at high elevations where oxygen pressure is low have higher haemoglobin concentrations, reduced oxygen saturation and an increased production of red blood cells to ensure oxygen supply to tissues. These physiological characteristics would result in identifying fewer cases of anaemia using the cutoff values in Table 3.2. The approach shown in Table 3.4 adjusts everyone’s haemoglobin value first, then applies the haemoglobin cutoff value for anaemia from Table 3.2.

The distribution of haemoglobin among sub-groups of the population can also provide important information concerning the aetiology of anaemia (vitamin A deficiency, iron deficiency, other nutrient deficiencies, inflammation status or blood disorders) ¹². For example, if the iron deficient population has the same distribution of haemoglobin as the iron replete population, then iron deficiency is unlikely to be the cause of anaemia. On the other hand, if the iron deficient population has a lower haemoglobin distribution, then it is more likely that iron is a cause of anaemia in that population.

Understanding the aetiology of anaemia is important for the design and evaluation of anaemia prevention strategies and programmes, thus a micronutrient survey should assess some of the factors in the above list. For example, in all malaria endemic countries, malaria should be assessed. Rapid diagnostic tests cost very little (around US$ 1) and are easy to ad#minister. Many resources that explain the samples and analyses required for assessing malaria and other infections, such as helminths, are available from WHO ^13,14,15.

Table 3.2. Haemoglobin cutoff values to define anaemia in individuals and populations (g/L) ^a

		Anaemiab
Population	Non-anaemiab	Mild	Moderate	Severe
Children 6-23 months	≥105	95-104	70-94	<70
Children 24-59 months	≥110	100-109	70-99	<70
Children 5-11 years	≥115	110-114	80-109	<80
Children 12-14 years, nonpregnant girls	≥120	110-119	80-109	<80
Children 12-14 years, boys	≥120	110-119	80-109	<80
Non-pregnant women (15-65 years)	≥120	110-119	80-109	<80
Pregnant women - First and Third Trimester	≥110	100-109	70-99	<70
Pregnant women - Second Trimester	≥105	95-104	70-94	<70
Men (15-65 years)	≥130	110-129	80-109	<80

^aSource: reference ^11
bHaemoglobin in grams per litre.

Table 3.3. Classification of public health significance of anaemia at the population level based on estimated prevalence of low haemoglobina^a

Category of public health significance	Prevalence of anaemia (%)
Severe	≥40
Moderate	20.0-39.9
Mild	5.0-19.9
Normal	≤4.9%

^aSource: reference¹¹.

Table 3.4. Haemoglobin adjustment for elevation and cigarette smoking^a

>

Elevation (metres above sea level)	Adjustment to individual haemoglobin value (g/L)
1-499	0
500-999	4
1000-1499	8
1500-1999	11
2000-2499	14
2500-2999	18
3000-3499	21
3500-3999	25
4000-4499	29
>4500	33
Cigarettes smoked per day
Non-smoker	0
Smoker, quantity unknown	3
under 10	3
10-19	5
>20	6

^aSource: reference¹¹.