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.

  1. Acute phase proteins are markers of subclinical inflammatory response that can affect interpretation of biomarkers, such as for iron and vitamin A status. 

  2. WHO guidelines on drawing blood: best practices in phlebotomy. Geneva: World Health Organization; 2010 (https://apps.who.int/iris/handle/10665/44294, accessed 20 March 2020).  2

  3. Note that this method does not determine the iodine status of the individual. 

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