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Laboratory assessment of folate (vitamin B9) status
  1. Agata Sobczyńska-Malefora1,2,
  2. Dominic J Harrington1,3
  1. 1 Nutristasis Unit, Viapath, St. Thomas’ Hospital, London, UK
  2. 2 Faculty of Life Sciences and Medicine, King’s College London, London, UK
  3. 3 Division of Women’s Health, School of Medicine, King’s College London, London, UK
  1. Correspondence to Dr Dominic J Harrington, St Thomas’ Hospital NHS Foundation Trust, London SE1 7EH, UK; Dominic.Harrington{at}


Folate (vitamin B9) plays a crucial role in fundamental cellular processes, including nucleic acid biosynthesis, methyl group biogenesis and amino acid metabolism. The detection and correction of folate deficiency prevents megaloblastic anaemia and reduces the risk of neural tube defects. Coexisting deficiencies of folate and vitamin B12 are associated with cognitive decline, depression and neuropathy. Folate deficiency and excess has also been implicated in some cancers. Excessive exposure to folic acid, a synthetic compound used in supplements and fortified foods, has also been linked to adverse health effects. Of at least three distinct laboratory markers of folate status, it is the total abundance of folate in serum/plasma that is used by the majority of laboratories. The analysis of folate in red cells is also commonly performed. Since the folate content of red cells is fixed during erythropoiesis, this marker is indicative of folate status over the preceding ~4 months. Poor stability, variation in polyglutamate chain length and unreliable extraction from red cells are factors that make the analysis of folate challenging. The clinical use of measuring specific folate species has also been explored. 5-Methyltetrahydrofolate, the main form of folate found in blood, is essential for the vitamin B12-dependent methionine synthase mediated remethylation of homocysteine to methionine. As such, homocysteine measurement reflects cellular folate and vitamin B12 use. When interpreting homocysteine results, age, sex and pregnancy, specific reference ranges should be applied. The evaluation of folate status using combined markers of abundance and cellular use has been adopted by some laboratories. In the presence of discordance between laboratory results and strong clinical features of deficiency, treatment should not be delayed. High folate status should be followed up with the assessment of vitamin B12 status, a review of previous results and reassessment of folic acid supplementation regime.

  • folate
  • vitaminB9
  • homocysteine
  • folic acid
  • diagnosis

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Folate is essential for the prevention of a wide spectrum of health issues, most notably megaloblastic anaemia and neural tube defects. Chronic suboptimal folate states may increase the risk of cognitive decline, depression, neuropathy and some cancers. However, this is often overlooked. The folate status of patients following cessation of treatment for previously identified deficient states is rarely monitored. There is also a poor awareness of the use of functional markers of folate status and of the possible negative consequences of excessive folic acid intake. Since vitamin B12 is essential for the transformation of 5-methyltetrahydrofolate (5-MTHF) to tetrahydrofolate (THF) for DNA synthesis, folate status assessment should not be considered in isolation. Here, we highlight the advantages and disadvantages of diagnostic markers of folate status, and make recommendations for the application and interpretation of laboratory tests in the clinical setting.


Folate metabolism and absorption

The term folate refers to derivatives of pteroylglutamic acid that drive major cellular processes, including nucleic acid biosynthesis, amino acid metabolism and methyl group biogenesis. These compounds consist of a pteridine ring linked to p-aminobenzoic acid and l-glutamic acid residues. Metabolic use is conferred through reduction of the pteridine ring to dihydrofolate (DHF) by dihydrofolate reductase (DHFR). Cellular DHF is further reduced to THF and conjugated to a polyglutamate chain. THF accepts one carbon unit from serine to form 5,10-methyleneTHF. In the cytoplasm, 5,10-methyleneTHF transfers its methylene group to deoxyuridine monophosphate, to synthesise deoxythymidine monophosphate, which is used for DNA biosynthesis and repair. 5,10-MethyleneTHF is oxidised to 5,10-methenylTHF, which in turn is converted to 10-formylTHF for purine synthesis. Methylenetetrahydrofolate reductase (MTHFR) is required for the conversion of 5,10-methyleneTHF to 5-MTHF - the form of folate necessary for remethylation of homocysteine to methionine via the vitamin B12-dependent methionine synthase (MS) enzyme.1

In nature, folate consists of a mixture of reduced folate polyglutamates. At physiological concentrations, absorption is reliant on hydrolysis to monoglutamate forms before transportation to jejunum mucosal cells by a saturable carrier-mediated process. Some passive diffusion also occurs at pharmacological concentrations.2 Absorbed folate monoglutamates are converted to 5-MTHF during transit through the intestinal mucosa before reaching the hepatic portal vein. 5-MTHF enters the plasma via the liver and is taken up by cells via a reduced folate carrier or through receptor-mediated endocytosis. The retention of folate by cells is dependent on conversion to its polyglutamate derivatives. 5-MTHF and folic acid are poor substrates for this conversion.3

Folate deficiency and excess

Poor dietary intake is the most common cause of folate deficiency. Increased folate turnover, that is, in pregnancy, breast feeding, skin disease (eg, psoriasis) and haematological disorders, can also lead to folate deficiency. Other major causative factors include conditions that confer gastrointestinal malabsorption. Anticonvulsants, folate antagonists, that is, methotrexate, or oral contraceptives also contribute to the development of folate deficiency.4 Inborn errors in folate metabolism lead to severe folate deficiency. Furthermore, folate deficiency is the most common cause of elevated plasma concentrations of total homocysteine (tHcy), a risk factor for cardiovascular diseases and dementia.5–7

One of the most important roles of folate in the prevention of disease was demonstrated when periconceptional folic acid supplementation was shown to guard against spina bifida.8–10 Routine periconceptional folic acid supplementation began,11 and mandatory fortification of enriched grain products followed in many countries.11 12 This initiative led to a significant reduction in the prevalence of neural tube defects13 and to increased serum folate concentrations across whole populations.14 The incidence of folic acid intakes that exceeded the upper tolerable limit of 1 mg/day also increased15 and unmetabolised folic acid (UMFA) was increasingly detected in serum.16 17 Although the clinical implications of the presence of UMFA, if any, have not been fully elucidated, excessive folic acid intake has been associated with adverse biochemical profiles and health effects. Most notably, the adverse effects were observed in the combination of high (defined as ≥45.3 nmol/L) folate with poor vitamin B12 status. For example, Miller et al found that such individuals had the highest concentration of tHcy and methylmalonic acid (a functional marker of B12 deficiency), and the lowest concentration of holotranscobalamin (the biologically active fraction of B12).18 Sawaengsri et al demonstrated that when folate intake was >2 times the Recommended Dietary Allowance (800 µg/day), individuals with the GG genotype of the 776C→G polymorphism of the B12 transport protein transcobalamin gene, which is associated with lower holotranscobalamin concentrations, had a 6.9-fold higher OR of neuropathy than those with the CC genotype.19 Likewise, Morris et al found that a combination of high serum folate and poor B12 status in elderly people predicted faster cognitive decline than normal folate with poor B12 status.17 In a study of the elderly in Chile, where a higher mandatory dose of folic acid is added to flour (220 µg/100 g) compared with the USA (80 µg/100 g), >26% of subjects had high serum folate (≥45.3 nmol/L), and a weaker biochemical response to a single B12 injection was observed than in those with serum folate ≥33.9 nmol/L.20 Moreover, some researchers suggest that the daily upper tolerable limit for folic acid of 1 mg/day is set too high as adverse neurological effects have been observed from long-term exposure to folic acid at doses of 0.5–1 mg in the presence of B12 deficiency.21 The combination of high folate and low B12 in mothers may also contribute to insulin resistance in the offspring22 and a higher risk of small-for-gestational-age infants.23 This is especially of relevance in countries such as India or Bangladesh where supraphysiological doses of folic acid (5 mg daily) are commonly prescribed during the first trimester to pregnant women.23

First-line assessment of folate status in the laboratory

Serum/plasma folate

The determination of folate in serum/plasma is the most common laboratory test used for folate status evaluation. Results mirror folate status closely for most patients and correlate with red cell folate (RCF) and tHcy. Serum/plasma folate concentrations rise in response to folic acid/folate ingestion for up to 2 hours and then decline rapidly. Fasting folate concentrations may represent reduced folate released by tissues.3 Results from non-fasting samples should be interpreted in the context of clinical symptoms (ie, pale skin, fatigue, lethargy, shortness of breath, mouth ulcers, tongue swelling), other available laboratory results (ie, full blood count) and previous serum/plasma folate results. Declining and suboptimal folate concentrations should be addressed since the risk of neural tube defects24 and the potential risk of certain types of cancers,25 Alzheimer’s disease and cardiovascular diseases26–28 increase in folate as well as B12 insufficiency.

The identification of individuals with unexpectedly high serum/plasma folate concentrations and coexisting B12 deficiency is important as correction of their folate and B12 status to adequate avoids a potential negative interaction on health. This is because patients with severe B12 deficiency often have impaired methionine synthesis and a corresponding accumulation of 5-MTHF, which diffuses out of cells resulting in a high serum/plasma folate concentration and low RCF concentration.29 5-MTHF is metabolically trapped in the absence of B12 and cannot be converted to THF thus conferring a functional folate-deficient state.30 The methyl-trap hypothesis explains why a severe B12 deficiency leads to megaloblastic anaemia, which highly resembles the syndrome seen in folate deficiency. Treating megaloblastic anaemia caused by B12 deficiency with folic acid produces only a marginal improvement. The newly available folate enables bone marrow cells to temporarily divide again before being trapped as 5-MTHF.

Analytical challenges associated with serum/plasma folate assays

Microbiological assays

Microbiological assays offer good sensitivity and have modest sample volume requirements. The use of 96-well microplates simplifies the assay and shortens analysis time.31 32 Yet, in comparison with folate-binding protein (FBP) assays, microbiological assays are laborious and require duplicate analyses to overcome poor precision. The growth medium is very rich and aseptic conditions are necessary to suppress growth of irrelevant micro-organisms.33 The development of folate-depleted cryoprotective preparations of Lactobacillus casei enabled improved reproducibility and stability of the organism for at least 8 months at −18°C.34 One also should keep in mind that the presence of antibiotics in blood samples may interfere with the assay. This issue was partially circumvented by the introduction of the chloramphenicol-resistant strain of L. casei. 35

Folate-binding proteins assays

FBP-based assays are the most commonly used method for the assessment of folate status. Labelled folate standard competes with endogenous folate for a folate binder, or in non-competitive binding assays, an excess of FBP is added to a sample followed by a labelled folate conjugate. Both set-ups are inexpensive and offer a quick analysis of large number of samples, plus a high degree of automation. Among the drawbacks, we can mention the different affinities that various folate forms have for FBP. Folic acid generally has a higher affinity for folate binders than 5-MTHF; however, at pH 9.3, the affinity of both compounds to FBP is equal.3 Because folic acid is more stable than 5-MTHF, it is often used as the assay calibrator. Yet, 5-MTHF is the predominant folate form in serum and red cells. Therefore, differences in FBP affinities generate misleadingly low results if assay conditions are not optimised.

The linear concentration range for FBP assays is narrow (~3.4–45.3 nmol/L). In the authors’ laboratory, 4% of samples analysed for serum/plasma folate exceed the upper margin of 45.3 nmol/L.36 Dilution of samples is problematic, as FBP assays are affected by medium composition, and unless samples are diluted with human albumin or folate free serum, the accuracy of folate results is diminished.

Based on UK National External Quality Assessment Scheme (NEQAS) Haematinics reports, Roche Cobas (USA), Abbott Architect (USA), Siemens Centaur (Germany) and Beckman Dxl (USA) are the most commonly used platforms for measuring serum/plasma folate. Other platforms include Beckman Access, Siemens Immulite 2000, Siemens Dimension/Vista and Ortho Vitros (USA). The interlaboratory CV for the same method and all methods are <10% and ~10%, respectively.

Second-line assessment of folate status in the laboratory

Red cell folate

The concentration of folate in red cells is a strong indicator of folate status because it is not influenced by transient changes in dietary intake. The concentration of folate in red cells is much higher than in plasma, and it is set at the time of cell production. Results provide an integrated average index, representing a 4-month period.37 Low values strongly suggest folate deficiency.

Analytical challenges associated with red cell folate analysis

Despite many advantages, the analysis of RCF is challenging because of (1) the chemical instability of folate, (2) the different lengths of the polyglutamate chains and (3) the often inefficient extraction of folate from cells. All RCF assays require the conversion of folate polyglutamates to monoglutamates. Incomplete conversion leads to higher results because folypolyglutamates have a higher affinity for FBP. Since the comparability of RCF methods is very poor, laboratories should establish their own reference intervals to get clinically useful results.

Total plasma homocysteine

tHcy is a sensitive marker of folate status that correlates well with serum folate and RCF.38 However, its specificity is compromised in patients with deficiencies in vitamins B2, B6 or B12, as well as in the cases of inborn errors/polymorphisms in genes related to homocysteine metabolism that is, MTHFR, MS and cystathionine-β-synthase (CBS). Approximately 1% of the population are heterozygous for CBS deficiency, which is associated with mild elevations in tHcy.39 A much more frequent polymorphism, affecting 5%–15% of the population, is caused by a MTHFR gene variant (C677T), which encodes thermolabile MTHFR. Individuals with this polymorphism have diminished MTHFR activity40 and a ~25% increase in tHcy concentration when compared with unaffected individuals. Age, gender, smoking, high alcohol and coffee consumption, sedentary lifestyle, stress, hormonal changes, drugs or clinical conditions affect homocysteine metabolism and its blood concentration.41 Men tend to have higher concentrations than women and tHcy is lower during pregnancy.42

Elevated tHcy concentration has been associated with numerous adverse health effects, most notably with cardiovascular diseases.43–45

Homocysteine methodologies

High-performance liquid chromatography (HPLC), immunoassays and liquid chromatography–tandem mass spectrometry (LC-MS/MS) are the three most popular techniques used for the measurement of tHcy. In the European Research Network for evaluation and improvement of screening, Diagnosis and treatment of Inherited Disorders of Metabolism (ERNDIM) proficiency testing scheme for tHcy (n=97 participants), 44% use LC-MS/MS; 18%, immunoassays; 15%, HPLC; and 12% use other methods (March 2018).

Analytical advantages and challenges associated with selected homocysteine assays

High-performance liquid chromatography

HPLC methods for the determination of tHcy have the advantage that other thiols can be simultaneously measured. As with other methods, the reduction of disulfide bonds and protein-bound homocysteine is required to free homocysteine. The extent of sample preparation varies according to the method of detection. HPLC methods are inexpensive when compared with the reagent costs of immunoassays and with the instrument costs of LC-MS/MS, yet they are more labour intensive and can process only a modest number of samples within a single analytical run.


Immunoassays for tHcy provide fast and high-throughput analysis and are the methods of choice in laboratories with a high sample turnover, or laboratories lacking access to LC-MS/MS. The main caveat associated with these methods is the high cost of reagents (eg, patented antibodies) and a limited dynamic range, set at 50 µmol/L. Nexo et al demonstrated good performance for fluorescence polarisation (FPIA) and enzyme-linked (EIA) immunoassays with a bias of −2 to +3% and +2 to +4% for FPIA and EIA, respectively, compared with gas chromatography mass spectrometry and HPLC methods. Both methods used the calibrators manufactured by AXIS (UK).46 A study by La’ulu et al showed a good correlation (r=0.95–0.99) between most of the tested immunoassays and HPLC.47

Liquid chromatography–tandem mass spectrometry

LC-MS/MS methods offer excellent sensitivity and specificity.48 49 Although the initial capital cost of LC-MS/MS instruments is high, reagent costs are relatively low and high-throughput analyses are possible. Yet, matrix effects may amplify the background noise and compromise sensitivity. Also, salts and detergents present in biological samples frequently cause ion suppression.

Third-line assessment of folate status in the laboratory

Individual folate forms in serum and red cells

The clinical use of determining individual forms of folate has been explored.50–52 Individuals with the MTHFR 677TT genotype have lower 5-MTHF as a consequence of a diminished capacity to convert 5,10-methyleneTHF to 5-MTHF. These individuals also tend to have a greater proportion of folate in the 5,10-methyleneTHF, THF and formyl folate forms when compared with CT and CC genotypes.50 53 Suboptimal 5-MTHF availability leads to an increase in tHcy, which has been associated with many diseases including cardiovascular diseases.43 44 There is also evidence suggesting that 5-MTHF deficiency may be a cardiovascular risk factor independent of homocysteine.54 55

Measurements of other forms of biologically active folate are currently poorly used in diagnostic settings. The development of the first isotope-dilution LC-MS/MS method for the simultaneous determination of 5-MTHF, THF, 5,10-methyleneTHF, methenylTHF/10-formylTHF and formylTHF offered a breakthrough in the assessment of the various folate forms.56 An automated 96-well plate assay was recently introduced to this method.50 It offered an excellent linearity in the range 0.22–220 nmol/L, providing a good reference method for the assessment of serum/plasma folate assays.57

Folic acid

The analysis of folic acid has gained additional interest after reports about the UMFA in people consuming fortified foods and folic acid supplements,58 and reported correlations with adverse health effects.59 60 Although this test has yet to be used by clinical laboratories, it has been included in the National Health Nutrition and Examination Survey (NHANES) population study.61–63 UMFA appears in serum if folic acid intake is higher than 200 µg/meal, which exceeds the capacity of the DHFR enzyme to reduce it to THF.64 The concentration is typically <2 nmol/L, but values as high as 278 nmol/L have been reported in healthy individuals consuming 5 mg of folic acid daily for 90 days.65

5-Methyltetrahydrofolate in cerebrospinal fluid

5-MTHF measurement in cerebrospinal fluid (CSF) has been used for the diagnosis and monitoring of cerebral folate deficiency (CFD), a neurological condition associated with low concentrations of 5-MTHF in the CSF and normal concentrations of serum/plasma folate/5-MTHF.66 A number of causes of CFD have been suggested, and among them we can mention the decreased transport across the blood–brain barrier caused by autoantibodies blocking folate receptor 1 (FOLR1) and mutations in the FOLR1 gene.67 Treatment with folinic acid (leucovorin or 5-formylTHF) appeared to be successful.66 68

Other markers of folate status

Several other potential markers of folate status have been suggested: urinary folate and folic acid; serum and urinary para-aminobenzoylglutamate and para-acetamidobenzoylglutamate; DNA methylation; uracil misincorporation into DNA and micronuclei; plasma and urinary formiminoglutamate and aminoimidazole carboxamide riboside.3 33

A 24-hour urinary folate/folic acid test provides unique information when performed in combination with serum and RCF.3

Global DNA methylation correlates well with serum/plasma folate and is potentially a sensitive tool for the identification of folate depletion.5 69 A low folate diet (50–120 µg of folate per day for 7–9 weeks) induced a detectable hypomethylation of DNA in peripheral blood mononuclear cells, whereas folate repletion over several weeks reversed the adverse effects.70 71 Jacob et al suggested that ‘normal’ folate concentrations (>7 nmol/L) are borderline at best and probably too low to maintain DNA methylation since tHcy begins to rise before the concentration of plasma folate falls into a true deficient state.70 Since rises in tHcy appear earlier than in DNA hypomethylation69 and because of complexity of the analysis, DNA methylation is unlikely to be used as a marker of folate status in a practical diagnostic setting.

Sample requirements for markers of folate status and sample stability

Serum/plasma folate

Samples should be collected into serum or lithium-heparin tubes following a 3-hour fasting, whereupon serum/plasma should be promptly separated to avoid haemolysis. EDTA tubes have also been used for collection, but may cause 5-MTHF to degrade at an estimated rate of ~2% per hour at room temperature.3 72

The analytes should also be protected from a direct light to avoid folate degradation. Some authors have, however, demonstrated that folate is relatively stable under ambient light and decays by 1.7% over 7 days, compared with 1% in the samples stored in dark conditions.73

In general, if testing is not to be performed within 2 days, samples for serum/plasma folate may be stored for 7 days at 2°C–8°C or 30 days at −20°C. Samples are stable for many years when kept at −70°C.

Red cell/whole blood folate

Whole blood EDTA non-fasting samples are used for RCF/whole blood analysis. The haematocrit should be determined prior to sample pre-treatment. Lysates are usually prepared with ascorbic acid within 2 days from blood collection using locally defined protocols. Ideally, samples should not be frozen before lysate preparation. UK NEQAS Haematinics reported that storage conditions of samples prior to RCF analysis vary greatly: 48% of laboratories store whole blood at 4°C and prepare lysates just before analysis; 26% prepare lysates on receiving the samples and freeze them at −20°C until analysis; 13% freeze the whole blood at −20°C and prepare lysates before analysis; 13% use other methods.74

Haemolysates with ascorbic acid are stable for several weeks at −20°C and several years at −70°C.3 At least three freeze/thaw cycles do not affect folate concentration in haemolysates. Freezing/thawing of undiluted whole blood samples should be avoided as this leads to significant folate degradation.

Most FBP assay kit manufacturers recommend protection of the whole blood samples from light and their processing within 2 days of collection. Stability studies have shown that if these conditions are not met, the whole blood specimens may still be used for the RCF analyses when using the Architect analyser.75

Cerebrospinal fluid 5-methyltetrahydrofolate

CSF should be collected into universal tubes, protected from light and immediately frozen at −70°C until analysis. The authors’ stability studies (unpublished data) of samples kept at 4°C demonstrated 25%, 32% and 43% of 5-MTHF degradation after 2, 3 and 4 weeks, respectively. Three freeze/thaw cycles did not affect 5-MTHF concentrations, leading to only 5% of decrease in concentration.

Total plasma homocysteine

Blood samples for tHcy analysis should be collected following an overnight fasting. A large protein meal can increase tHcy concentrations by 10%–15% after 6 to 8 hours.76 Factors affecting albumin concentrations also alter tHcy measurements since homocysteine is protein bound, and venepuncture should not be performed after venous stasis or following the subject resting in a supine position. SST, EDTA and lithium-heparin tubes are often used for tHcy analysis. If citrated plasma is used, the result should be multiplied by a factor of 1.1 to correct for the volume of citrate in the tube. Plasma should be separated from red cells within 60 min and kept on ice prior to separation. Delayed removal of red cells results in artificially high tHcy concentrations because of homocysteine release from blood cells; tHcy increases by 1 µmol/L in 3 hours if the sample is kept at room temperature.3 After separation, plasma is stable for at least 4 days at room temperature, several weeks in the fridge and for several years at −20°C.77

Alternatively, samples can be collected into tubes containing a preservative, eg Kabevette Vacuum HCY 837 V 3.5 (Kabe Labortechnik GmbH). Kabevette tubes prolong the stability of unspun samples for up to 72 hours.78

Interpretation of markers of folate status: reference ranges

Serum/plasma and red cell folate

WHO initially proposed cut-offs for folate deficiency based on the concentration at which macrocytic anaemia is likely to appear (<6.8 nmol/L for serum/plasma folate and <227 nmol/L for RCF).79 80 These cut-offs were endorsed in the subsequent WHO consultations.81 82 The cut-offs were later revised to reflect folate deficiency based on the functional indicators.83 Irrespective of age and gender, and after adjustment for B12 and creatinine, the relationship between folate and logarithmic concentration of homocysteine is biphasic (if using linear approximations): at low folate concentrations, tHcy concentrations increase as folate concentration falls, but at higher folate concentrations, homocysteine remains unchanged (or follows a line with a gentler slope). Based on this model, the concentration below which tHcy begins to rise is 10 nmol/L for serum folate/plasma and 340 nmol/L for RCF. The authors stated that the value of 340 nmol/L for RCF cannot be applied to expectant mothers because this concentration is not adequate enough for the prevention of neural tube defects.83 In 2015, WHO made a recommendation for the determination of optimal folate status (>906 nmol/L) for women of reproductive age. Other cut-offs in common use are those recommended by the Institute of Medicine: 7 nmol/L for serum/plasma and 305 nmol/L for RCF.84

High folate status has been arbitrarily defined as serum/plasma folate ≥45.3 nmol/L.80 This value also represents the highest limit of the linear range for many FBP assays. Other cut-offs used to define high folate status in studies include 59 nmol/L,85 33.9 nmol/L20 or 30 nmol/L.86 The diagnostic follow-up of high folate results is rarely carried out.

Cut-offs assigned by different folate methodologies may not be transferrable between methods. Methodology bias is especially pronounced for RCF assays. As a result, laboratories should establish their own reference ranges. The UK NEQAS Haematinics assay survey (2007/2008) showed a great diversity in the lower and upper limits of reference ranges for serum/plasma and RCF assays (both within the same, and between different methodologies): 40% of laboratories established reference ranges, 30% used data supplied by kit manufacturers, 22% used historical data and 8% adopted ranges from other laboratories using the same instrumentation or employed ranges from the published literature.74

Unmetabolised folic acid

The reference range for UMFA is yet to be established as the debate continues regarding the safe use of folic acid compounded with the duration of its intake. In their study, Paniz et al defined high UMFA as >1.12 nmol/L, based on the 95th percentile in the distribution of values obtained from 1730 young adults (aged 20–39 years) in the NHANES study.65 87 However, Pfeiffer et al observed a significant difference in UMFA between fasted and non-fasted subjects. The central 95% reference intervals for UMFA were 0.383 to 4.93 and 0.45 to 28.0 nmol/L for fasted and non-fasted subjects (N total=~7500, aged ≥1 year).87

Total plasma homocysteine

The most commonly used upper reference range limit for tHcy is 15 µmol/L.88 Other cut-offs in use include 10 μmol/L,89 12 μmol/L,90 14 μmol/L,91 16 μmol/L92 and 18 μmol/L. Adult men typically have a tHcy concentration ~2 μmol/L higher than women.52 93 Since tHcy is strongly influenced by age, sex and pregnancy, specific upper limits should be applied. Suggested cut-offs include <10 µmol/L for children and pregnant women; <15 μmol/L, for adults; <20 μmol/L for those >65 years.77 The pregnancy-related cut-offs may be further categorised by trimesters. Lower tHcy concentrations in the first trimester reflect the intake of folic acid and haemodilution, while higher tHcy concentrations in the third trimester are reflective of lowering of B12 status.94 tHcy 3.7–6.9 (first), 2.7–7.2 (second) and 2.4–9.6 (third trimester) µmol/L are examples of pregnancy-specific reference ranges recently established by the authors.95

Calibration traceability

External quality assurance (EQA) schemes in which performance is based on consensus are available for serum/plasma folate and RCF measurement. Comparing results with a higher-order reference procedure and analysing standard reference material may provide more reassurance in terms of achieving accurate folate results. Isotope dilution LC-MS/MS assays are accepted as reference methods for the quantification of serum folate.57 Using this method as a reference, UK NEQAS Haematinics assessed the accuracy of all other methods, finding the best performing method to be traceable to the WHO 03/178 standard.57

UK NEQAS Haematinics scheme for serum folate reports an intramethod CV of 5%–12%, with overall CVs for all methods being <10%. However, significant variability exists for RCF, with intramethod CVs of 12%–32% and overall CVs ~40% (UK NEQAS Haematinics, Nov 2017).

Homocysteine assays generally demonstrate good performance and several EQA schemes are available.

Serum-based international reference materials are available but only provide certified concentrations for 5-MTHF and tHcy. NIST developed SRM 1955 and SRM 1950. SRM 1955 has been available since 2006 and provides three certified values for 5-MTHF and tHcy and reference values for folic acid, 5-formylTHF and total folate. SRM 1950 uses human frozen plasma, has one level only and, like SRM 1955, gives certified values for 5-MTHF and tHcy, and a reference value for folic acid. The UK-based National Institute for Biological Standards and Control and the WHO International Laboratory for Biological Standards have developed 03/178. One level of a freeze-dried specimen is provided and reference values for 5-MTHF, 5-formylTHF and total folate are given. The same institution developed a whole blood folate international standard (95/528); freeze-dried human whole-blood haemolysate at one concentration with consensus values for total folate.

Example of laboratory assessment algorithm

An example algorithm for the assessment of folate status is shown in figure 1. When serum/plasma folate is within the possible deficiency range,80 tHcy concentrations should be checked, providing that the patient has normal renal function. RCF may be measured if tHcy measurements are inconclusive or compromised through impaired renal function. The assessment of B12 status should be performed in all patients with elevated tHcy. 5-MTHF and MTHFR analysis may be informative when seeking the cause of low folate and elevated tHcy. High folate status should be followed up with the assessment of B12 status, review of previous results and assessment of continued folic acid treatment/supplementation reduction. If relevant and available, UMFA testing may be performed.

Figure 1

Example of algorithm to assess the folate status. Dashed arrows indicate optional testing. 5-MTHF, 5-methyltetrahydrofolate; FBC, full blood count; holoTC, holotranscobalamin; MMA, methylmalonic acid; MTHFR, methylene tetrahydrofolate reductase. *WHO recommended.80 **Age, sex and pregnancy related, ie, <10 µmol/L for children <15 years and pregnant women,77 <13 μmol/L for women and <15 μmol/L for men aged 19–64 years52 and <20 μmol/L for elderly >65 years.77 ***An algorithm suggested by Harrington can be used.96


Low serum/plasma folate in the presence of clinical symptoms strongly suggests folate deficiency. Suboptimal folate status should be treated, especially if accompanied by elevated tHcy. Monitoring of folate status following cessation of folic acid supplementation is required to ensure adequate folate status. RCF testing may be helpful, providing that the appropriate cut-offs is used. High folate status should be reviewed and the adequacy of B12 confirmed.


Many thanks go to Kaiya Chowdhary of Nutristasis Unit for conducting red cell folate stability study and to Matthew Critcher from the University of Nottingham for his work with Kabevette tubes.


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  • Handling editor Tahir S Pillay.

  • Contributors Both authors contributed to this manuscript. AS-M produced the first draft from which the final edit for submission was produced together.

  • Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.

  • Competing interests None declared.

  • Patient consent Not required.

  • Provenance and peer review Commissioned; externally peer reviewed.

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