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Module 3 / Folate

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.

  1. 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. ↩

  2. Serum and red blood cell folate concentrations for assessing folate status in populations. Vitamin and Mineral Nutrition Information System. Geneva: World Health Organization; 2015 (WHO/NMH/NHD/EPG/15.01; https://apps.who.int/iris/bitstream/handle/10665/162114/WHO_NMH_NHD_EPG_15.01.pdf, accessed 14 June 2020). ↩ ↩2

  3. Drammeh BS, Schleicher RL, Pfeiffer CM, Jain RB, Zhang M, Nguyen PH. Effects of delayed specimen processing and freezing on serum concentrations of selected nutritional indicators. Clin Chem. 2008;54:1883–91. doi: 10.1373/clinchem.2008.108761. ↩ ↩2

  4. O’Broin SD, Gunter EW. Screening of folate status with use of dried blood spots on filter paper. Am J Clin Nutr. 1999;70:359–67. ↩ ↩2

  5. Pfeiffer CM, Sternberg MR, Hamner HC, Crider KS, Lacher DA, Rogers LM et al. Applying inappropriate cutoffs leads to misinterpretation of folate status in the US population. Am J Clin Nutr. 2016;104:1607–15. ↩ ↩2

  6. Rogers LM, Cordero AM, Pfeiffer CM, Hausman DB, Tsang BL, De-Regil LM et al. Global folate status in women of reproductive age: a systematic review with emphasis on methodological issues. Ann N Y Acad Sci. 2018;1431:35–57. doi: 10.1111/nyas.13963. ↩

  7. Guideline: Optimal serum and red blood cell folate concentrations in women of reproductive age for prevention of neural tube defects. Geneva: World Health Organization; 2015 (https://apps.who.int/iris/bitstream/handle/10665/161988/9789241549042_eng.pdf, accessed 14 June 2020). ↩ ↩2 ↩3 ↩4

  8. Zhang M, Sternberg MR, Yeung LF, Pfeiffer CM. Population RBC folate concentrations can be accurately estimated from measured whole blood folate, measured hemoglobin, and predicted serum folate-cross-sectional data from the NHANES 1988-2010. Am J Clin Nutr. 2020;111:601–12. doi:10.1093/ajcn/nqz307. ↩

  9. Biological reference materials [website]. Hertfordshire: National Institute for Biological Standards and Control (NIBSC); 2020 (https://nibsc.org/products/brm_product_catalogue.aspx, accessed 14 June 2020). ↩ ↩2

  10. Proficiency testing [website]. Northfield (IL): US College of American Pathologists (CAP); 2020 (https://www.cap.org/laboratory-improvement/proficiency-testing, accessed 14 June 2020). ↩ ↩2

  11. Vitamin B12, folate, ferritin, red cell folate. Clinical Chemistry [website]. Birmingham: UK NEQAS; 2020 (https://ukneqas.org.uk/programmes/result/?programme=vitamin-b12%2C-folate.-ferritin%2C-red-cell-folate, accessed 14 June 2020). ↩

  12. Performance Verification Program for Serum Micronutrients [website]. Atlanta: US Centers for Disease Control and Prevention (CDC); 2019 (https://www.cdc.gov/nceh/dls/nbb_micronutrient_performance.html, accessed 14 June 2020). ↩ ↩2 ↩3

  13. Quality control materials for serum micronutrients [website]. Atlanta: US Centers for Disease Control and Prevention (CDC); 2019 (https://www.cdc.gov/nceh/dls/nbb_micronutrient_materials.html, accessed 11 May 2020). ↩ ↩2

  14. Haynes BM, Pfeiffer CM, Sternberg MR, Schleicher RL. Selected physiologic variables are weakly to moderately associated with 29 biomarkers of diet and nutrition, NHANES 2003-2006. J Nutr. 2013;143:1001S–10S. doi: 10.3945/jn.112.172882. ↩

  15. Zhang DJ, Elswick RK, Miller WG, Bailey JL. Effect of serum-clot contact time on clinical chemistry laboratory results. Clin Chem. 1998;(6 Pt 1):1325-33. ↩

  16. National Health and Nutrition Examination Survey (NHANES) laboratory procedure manual, 2011-12: RBC folate in whole blood. Atlanta: US Centers for Disease Control and Prevention; 2011. ↩

  17. de Benoist B. Conclusions of a WHO Technical Consultation on folate and vitamin B12 deficiencies. Food Nutr Bull. 2008;29(2 Suppl):S238-44. ↩