Module 2. Indicators of programme coverage, specimen selection, management and analysis

Assessing indicators of programme coverage

Collecting specimens for assessing biomarkers

Location for collecting samples and data

Selecting laboratories to test specimens

Introduction

The overall objectives of the micronutrient survey describe the populations to be included, as well as the micronutrients and programme process indicators to be assessed (see Module 1, Planning and designing a micronutrient survey). The subsequent steps determine the type of samples and specimens required, and how they will be collected, transported, stored and analysed. Development of recommendations concerning most biomarkers ¹ and food nutrients requires analysis in a laboratory setting with specialized equipment and training. These are described in more detail in Module 3: Biomarker selection and specimen handling.

Decisions made during the planning process will influence the survey outcome, protocol development, draft budget, and equipment and supplies needed. Some points to consider:

• program coverage indicators will require knowledge of national and large-scale nutrition interventions, and may reflect a national nutrition policy;
• the selection of indicators will be based on the feasibility of collecting required food samples and biological specimens, maintaining a cold chain if required, access to appropriate laboratory capacity either in the country or internationally, and available budget; and
• the survey scope may need to be adjusted, depending on the cost of collecting, transporting, and analysing the samples and specimens required to assess the chosen indicator(s).

Module 3: Biomarker selection and specimen handling describes the options and main factors to consider in making these decisions.

Ordering equipment and supplies is an important determinant of the survey timeline, as described further in Module 9, Survey equipment and supplies and should be done well in advance of the survey. At the same time, if this is done too early there is a risk of exceeding expiration dates on the supplies purchased (for example, cuvettes for a portable photometer).

Introduction

The overall objectives of the micronutrient survey describe the populations to be included, as well as the micronutrients and programme process indicators to be assessed (see Module 1, Planning and designing a micronutrient survey). The subsequent steps determine the type of samples and specimens required, and how they will be collected, transported, stored and analysed. Development of recommendations concerning most biomarkers ¹ and food nutrients requires analysis in a laboratory setting with specialized equipment and training. These are described in more detail in Module 3: Biomarker selection and specimen handling.

Decisions made during the planning process will influence the survey outcome, protocol development, draft budget, and equipment and supplies needed. Some points to consider:

• program coverage indicators will require knowledge of national and large-scale nutrition interventions, and may reflect a national nutrition policy;
• the selection of indicators will be based on the feasibility of collecting required food samples and biological specimens, maintaining a cold chain if required, access to appropriate laboratory capacity either in the country or internationally, and available budget; and
• the survey scope may need to be adjusted, depending on the cost of collecting, transporting, and analysing the samples and specimens required to assess the chosen indicator(s).

Module 3: Biomarker selection and specimen handling describes the options and main factors to consider in making these decisions.

Ordering equipment and supplies is an important determinant of the survey timeline, as described further in Module 9, Survey equipment and supplies and should be done well in advance of the survey. At the same time, if this is done too early there is a risk of exceeding expiration dates on the supplies purchased (for example, cuvettes for a portable photometer).

Assessing indicators of programme coverage

Indicators of large-scale interventions to improve micronutrient status are often included in a micronutrient survey. This allows the investigation of the relationship between micronutrient status and access to interventions. Results can be applied to improve programmes and policies, as well as to understand impact. Nationally relevant intervention indicators should be incorporated and can include questions about the knowledge and use of fortified foods, micronutrient supplements, and point-of-use fortification (for example, micronutrient powders, or MNP) by the intended user group.

Coverage of interventions such as vitamin and mineral supplementation, point-of-use fortification and deworming can be assessed in a micronutrient survey, in a variety of ways. The most common way to obtain information is via self-reporting. Some questions, for example, if or when a particular health service was received, can be validated through examination of medical records, child health cards or immunization records. The documentation varies by country, and each type has its limitations. Documents may not be completed reliably to reflect the true coverage or timing of the intervention of interest, especially when the intervention is part of a mass campaign where the priority is to deliver interventions to the largest possible number of eligible participants in a given time period. Self-reporting by adults, and caregiver recall for children, are subject to several biases including socially desirable reporting and potential errors associated with long recall periods. Recognizing this, coverage data are often collected using different sources within a single survey. These data are then reported separately, compared, and triangulated with other data sources.

Coverage of vitamin and mineral supplementation

Two crucial issues to consider when collecting data on the provision or consumption of vitamin and mineral supplements are the potential for confusion between the objective of the supplements (prevention or treatment), and the timing of the survey. When assessing access to and coverage of a national supplementation programme, it is helpful to show the participant an example of the exact supplement.

Supplementation with vitamins and minerals is usually specific to certain population groups and, depending on the programme, the same supplement could be intended for prevention or for treatment. For example, iron-folic acid supplements might be provided periodically to all adolescent girls to prevent iron deficiency. However, the same or a different-looking supplement may be provided as treatment when an individual is clinically diagnosed with anaemia or iron deficiency. Similarly, it is common for children 6–59 months of age to receive high-dose vitamin A capsules every two years; however, clinicians may also administer capsules as part of routine care for acute illness. The questionnaire should include questions that differentiate between the use of supplements for prevention and as part of clinical care. Often the interviewer will ask to see the supplement bottle used by the participants, and is trained to record the micronutrients contained or the brand name shown on the label.

Questions requiring recall about supplements should be based on a time period that reflects the particular programme cycle and on evidence-based expectations for reliable recall. For reporting on the use of antenatal iron and folic acid supplementation (in tracking progress towards the Global nutrition target of reducing anaemia among women of reproductive age), it has been suggested to ask women about their consumption of iron-containing supplements during a current or past pregnancy within the last two years.¹ When assessing the coverage of high-dose vitamin A supplementation for children, the Global Alliance for Vitamin A (GAVA) recommends using data from post-event coverage surveys rather than from household surveys.² There is a transient increase in serum retinol following high-dose vitamin A supplementation. Thus, when assessing vitamin A status in a survey, it is important to time the survey so that a sufficient amount of time has passed since the last vitamin A supplementation campaign.

Coverage of point-of-use fortification

In many countries, point-of-use fortification (also called ‘home fortification’) is used to improve the nutritional status of children under 5 years of age or the status of school age children. This is most often done through the use of micronutrient powders (MNP). Depending on the scope of the intervention, the survey may include questions related to the use of MNP. Where MNP are provided in only a limited area of the country, the expected positive responses for their use may be too low to have relevance in the proposed survey.

When the use of MNPs is assessed as part of a micronutrient survey, questions are usually included on both receipt of the product and intake of MNP sachets by the intended population group. Other useful questions might concern the receipt of counselling on correct use of the MNP and the perceived benefits and side effects. An example of a module for MNPs can be found in the MNP questionnaire template online tool.

Coverage and quality of fortified or fortifiable foods

Many countries implement large-scale food fortification to achieve optimal intake of a range of micronutrients. Fortifiable foods generally refer to commonly consumed food items (usually staple foods or condiments) that can be produced on a large scale, meaning at least 20 metric tons per day, by companies with the technical and financial capacity to add micronutrients in line with voluntary or mandatory fortification regulations and policies. Examples of such foods include flours (usually wheat or corn), cooking oil, sugar, food-grade salt and other condiments (such as bouillon, soy sauce and fish sauce). It is common to include indicators of household use of foods that are fortified or fortifiable, as well as indicators of consumption among groups that are more vulnerable to certain deficiencies. There are two categories of foods to consider incorporating into surveys: those mandated to be fortified according to national policies and regulations, and those that are potentially fortifiable and are likely to reach the main population of interest. Relevant issues to consider when selecting programme indicators to collect on large-scale fortifiable foods are shown in Box 2.1.

Many surveys collect a small sample of the fortifiable food from households to test for the presence and concentration of micronutrients. This is most commonly done to assess household coverage of iodized and adequately iodized salt, but it can also be done with edible oil, flour and other products. The planning team may decide to provide a replacement sample or a small monetary contribution to the households. This can be particularly important in settings where not providing a replacement or contribution will affect response rates or may adversely affect the family, for example by leaving them with no oil for cooking for the day and no resources to get more. Food replacements or monetary contributions need to be included in the overall budget and field logistics plan.

The presence of iodine in salt has been routinely tested in the field using specially developed rapid test kits. However, these kits are not quantitative and do not provide reliable estimates of salt iodine concentration. Various methods are being explored for salt and other means that involve collecting and analysing individual and composite samples, with various qualitative and quantitative protocols to assess micronutrient content.

Comprehensive information on testing iodine in salt can be found in the UNICEF document Guidance on the monitoring of salt iodization programmes and determination of population iodine status. ³

Box 2.1. Factors to consider when deciding on the type and wording of programme indicators for large-scale fortifiable foods

• Do fortification regulations apply to only a specific type of the fortifiable food, or do typical industry practices mean that certain product types are more likely to be fortified?
  • If fortified wheat flour regulations are applied only to high extraction (“white”) flour or flour used to bake bread, then indicators need to be designed for that product type accordingly.
  • Salt iodization quality can vary widely by salt type. Where a range of salt grain types are consumed, it is necessary to distinguish the main type of salt used in a household. Where a sample is collected for quantitative assessment of iodine, laboratory personnel should also determine and record the salt type according to clear standards.
• Are there labeling or specific logo requirements for fortified products?
  • Observation of, or questions about food labels could be used to assess the use of a fortified product. For example, the national policy may require use of a specific logo in order to support demand and consumer choice to fortified foods. This means that logo or labeling information will also indicate compliance with national policies. However, not all products will be in their original packaging and some products may be falsely labeled.
• Is there extensive home production of the fortifiable product, or is it mainly commercially produced?
  • Home produced staple foods are generally not fortified, unless there was hammer mill level fortification. Therefore, a question about the source of the product is helpful so only the relevant data about commercially produced foods will be analysed and presented to determine industry compliance.
  • Questions on brand name, manufacturer, and country of origin are also useful for internal program management, although it is generally not acceptable to present nutrient content analyzed by a salt manufacturer in a publicly available report.
• Is (or was) there a communication component to the overall national fortification strategy?
  • If such a component exists or existed in the past, then consider including questions about respondent knowledge and awareness of the fortified product as well as the source of this information. In addition, including a question on whether the respondent looks for and identifies the fortified product at purchase may provide useful information for monitoring the communication component.
• Is the fortifiable staple product used by the food industry to produce processed foods or condiments with a large market across different consumer groups?
  • If so, then questions about consumption of these specific processed foods or condiments could be included in the questionnaire. For example, bouillon is a major source of (potentially iodized) salt in many West African countries, and subsidized Baladi bread is an important source of fortified flour in Egypt.

Supplies for collecting and storing food samples need to be considered in addition to those for biologic specimens. Generally, simple collection containers can be used—resealable plastic bags for dry food products and containers with screw tops for edible oil. Some foods, such as bread, involve additional logistical considerations because the bread must be weighed to assess water content at the point of collection and at the laboratory, and will become moldy within a few days or weeks. In general, food samples should be stored and transported to the laboratory in cool conditions and protected from direct sunlight.

Additional details on food sample collection techniques, storage and transport considerations, analytical methods and presentation of resulting data can be found in the “Analysis of Food Samples” online tool.

Collecting specimens for assessing biomarkers

The subclinical status for most micronutrients is assessed using biomarkers found in biological specimens (blood or urine). It is not current practice to include clinical signs and symptoms of micronutrient deficiencies or excess in large population-based surveys. Module 3: Biomarker selection and specimen handling, and the “Biomarker Selection and Specimen Handling” online tool provides detailed information.

Surveys can also include the collection of food samples for assessing specific nutrient levels. Discussions with experts during the planning stage should specify the type of specimens or samples needed for each biomarker and food nutrient, the most appropriate collection method and devices needed, processing, and requirements for transportation and storage. Other specimens may be collected for different purposes, such as stool specimens for assessing intestinal parasites. Detailed discussion of these specimens is beyond the scope of this manual.

Collecting blood specimens

Blood specimens are required for assessing the following common indicators:

• haemoglobin concentration (as an indicator of anemia)
• micronutrient status of populations: iron, vitamin A, vitamin D, vitamin B12, folate, and zinc
• acute phase proteins, ¹ primarily C-reactive protein (CRP) and α-1-acid glycoprotein (AGP)
• other factors related to measured micronutrient status, such as malaria or other infections, and, in countries where these conditions may be prevalent, hemoglobinopathies.

Blood specimens can be collected using either capillary or venous sampling, depending on the volume of blood needed for laboratory testing. The collection device and subsequent blood processing, if any, depend on the blood fraction required for the biomarker of interest. Blood fractions include red blood cells, plasma, and serum. Different collection tubes may be required to provide the types of specimen needed for different analyses (see Module 3: Biomarker selection and specimen handling). Collection of specimens as dried blood spots on filter paper is not currently recommended for assessing micronutrient status in cross-sectional surveys. The one exception is folate status assessment with a microbiologic assay.

Testing for haemoglobin levels (using a portable photometer) and for malaria (using a rapid diagnostic test) can be conducted in the field using small volumes (~30 µL) of capillary whole blood obtained from a finger or heel prick. Otherwise, biological specimens generally require collection, storage, and transport to a laboratory for processing and analysis.

Capillary Blood Collection

To collect a capillary blood sample from a finger or heel prick, use a disposable, single-use, contact-activated lancet. Lancets are available with different blade widths and depths. The more blood that is needed, the greater the blade depth and/or width is required.² The age of the population group also needs to be considered.

Capillary sampling can be used to collect blood into a small, trace element-free, blood collection tube (with or without anticoagulant) for laboratory assessment of a wide range of biomarkers. The maximum collected is usually 500 µL of capillary blood, an amount that generates approximately 200 µL of serum or plasma.

Survey staff with or without phlebotomy experience can be trained to collect capillary blood samples. However, it is essential that experienced trainers conduct the training and ensure the use of standardized techniques. If larger volumes of capillary blood samples are required, particularly from young children, extensive training is needed. Only trainees with demonstrated skills and confidence should be selected for these tasks.

Venous Blood Collection

Venous blood collection is used when the total blood volume needed exceeds what can be collected by capillary sampling. The volume needed depends on the number of laboratory tests and the types of assays used. For large-scale, population-based surveys, only experienced phlebotomists should collect venous blood specimens. It is particularly difficult to successfully collect venous blood from young children and from malnourished and dehydrated individuals. Therefore, in micronutrient surveys, it is best practice to select phlebotomists who perform this procedure on a regular basis, particularly among the selected population groups.

The exact supplies needed for blood collection depend on the tests to be performed. More details are available in Module 3: Biomarker selection and specimen handling and in the “Equipment and Supplies List (more complex worksheet)” online tool.

Blood processing in the field and managing a cold chain when collecting blood specimens

Capillary or venous blood analysis requires centrifugation, typically within 24 hours of collection. In addition, biological specimens usually require a cold chain that begins the moment a sample is collected and is functional through processing, storage, transport and arrival at the laboratory that will conduct the analysis. This avoids the risk that temperature changes (thawing and refreezing) alter the outcome of the analyses (see Module 3: Biomarker selection and specimen handling). Mobile freezers will be needed if the length of time between specimen collection and storage at a facility with a -20°C freezer is too long.

In some countries, facilities with reliable electrical power, centrifuges and −20°C freezers are available for survey teams to use for processing and storing specimens during fieldwork. However, if this is not the case, analysis teams may need a self-contained field laboratory. Field laboratory equipment usually includes portable centrifuges and −20°C portable freezers. Centrifuges may be powered by a car or motorbike battery and can be used for the timely processing of blood specimens into serum or plasma fractions, for storage in cryovials. Portable freezers may be powered by a car battery, a portable generator, or standard electrical outlets. It is useful to freeze gel packs when storing specimens in a cool box prior to processing and then to freeze the specimens quickly after processing to maintain the cold chain in the field. It is important to consider the space required in the field vehicles for −20°C portable freezers and other equipment and supplies, so that plans and budgets include the appropriate size and number of vehicles.

Some biomarkers require additional special considerations. For example, if assessment of serum or plasma zinc concentration is included in the survey plan, strict precautions must be taken to avoid contamination from exogenous sources, either in a temporary field laboratory or in a facility laboratory. You can find more information on the specific needs for each micronutrient test in Module 3: Biomarker selection and specimen handling.

Collecting urine specimens

A spot (single) urine specimen is recommended in cross-sectional surveys. These specimens are analysed in the laboratory for urinary iodine concentration (UIC), which can be used to determine population iodine status.³ In general, individuals are asked to capture urine in a disposable sterile cup from which the urine is then pipetted into small sterile cryovials. The urine specimens do not need to be kept cool for analytical purposes, but it is convenient to store them in the cool box with other specimens. This will also reduce odour. You can find additional details for urine collection for the assessment of iodine concentration in Module 3: Biomarker selection and specimen handling

Safety and other issues

When collecting blood and urine specimens there are a number of other issues to consider related to training, personal safety, hygiene, equipment, field processing and storage. These are discussed in the “IATA guidance document on infectious substances” online tool and in WHO guidelines on drawing blood.² For example, appropriate sharps and biohazard disposable containers and protocols must be available for the safe disposal of cuvettes, blood tubes, needles and lancets.

Location of collecting samples and data

The survey management team should determine where the interviews, the collection of biologic specimens and food samples and biological laboratory processing will take place. These may be at the same or different locations. With more complex surveys, especially those in settings where households are very far apart or in difficult terrain, it may be best to set up a mobile laboratory in a central location. Sampling could also be done in households or through a clinic or school.

The main advantages of setting up a field laboratory for collecting and processing data include:

• A minimum number of sets of equipment may be required per team, thus reducing costs.
• Data collection in a single place eliminates the time and effort needed to move between households carrying equipment and supplies, including anthropometry equipment, that would need to be set up in each household. It also reduces data collection time, is more convenient for the survey team members, prolongs equipment life and can control the conditions where precautions against contamination are required, such as with blood collection for zinc analysis.

However, it is not always possible to find a convenient central site or it might be inconvenient for participants to travel to the central location.

Conducting interviews at the household rather than in a central location can improve participant comfort and privacy. In addition, household-level data collection may lead to more complete information about programme processes, such as being able to access child health cards and observe the labeling on food products. A household location also helps avoid potential biases in the collection of food samples, for example, a respondent may buy or obtain some of the requested food on their way to the central site.

It may be useful to use both locations, for example asking individual questions and collecting food samples at the household, and collecting biological specimens and anthropometry data at a central location. No matter the setting, care must be taken to match the unique ID labels for questionnaires with associated samples and specimens so that they can be correctly linked during data management and analysis.

All field procedures must be pilot tested. The survey management team needs to be able to change plans based on the most efficient and effective method for high-quality data collection and high response rates. The selected method should be the same for each cluster.

Selecting laboratories to test specimens

Access to laboratories with high-quality performance and an acceptable cost per specimen is a critical factor in determining the feasibility of including selected vitamin or mineral biomarkers in a micronutrient survey. The laboratories may be national or international. If international, the survey management team needs to plan for the required export permissions and costs of shipping samples or specimens out of the country, as well as for the requirements for them to be accepted in the receiving country. Identification of laboratories to conduct quality-assured analyses should be done very early in the planning process.

Laboratories under consideration for micronutrient biomarker analysis should first be assessed by experts in the field. The assessment should consider the following criteria:

1. The laboratory performs analyses using accepted standard methods (usually the gold standard or an internationally accepted standard);
2. The laboratory staff has the technical skills required for performing the analyses;
3. The analyses are routinely carried out in the laboratory (within the last few months);
4. The laboratory has the ability to analyse the appropriate number of samples daily to provide the necessary throughput for a survey and maintains the instruments in good working condition;
5. Quality assurance measures are in place or can be easily set-up including:

• a. Preparing quality control (QC) materials for analysing survey samples
• b. Using bench QC pools for every run
• c. Using blind QC pools for every run
• d. The laboratory participates and performs well in external quality assurance (EQA) programmes, including the Centers for Disease Control and Prevention’s (CDC’s) Vitamin A Laboratory – External Quality Assurance (VITAL-EQA for vitamin A, B vitamins, vitamin D, iron and CRP) programme,¹ Ensuring the Quality of Iodine Procedures (EQUIP for iodine) programme,² the Vitamin D External Quality Assessment Scheme (DEQAS for 25-hydroxy-vitamin D),³ and EQA for the folate microbiological assay (serum and whole blood)
• e. Use of international reference material (where available) to periodically verify the accuracy of other methods used, and acceptable performance for all selected analytes.

It is preferable that a single laboratory analyses all specimens for a specific assay. This will avoid any possible inter-laboratory variation. The ”Questionnaire for laboratory evaluation” online form can assist with the selection process.

Key issues to consider when selecting a method include:

• the type of biological specimens and/or food samples required
• the number and volume of specimens and/or samples to be tested
• the timeline for completing tests (testing large numbers of specimens for certain nutrients may take several months)
• how the specimens should be collected, processed, stored, and transported to the laboratory (this will include any cold chain requirements)
• assay costs
• the requirements and costs of quality assurance processes for each method.