Vitamin B12 and folate tests: interpret with care

Vitamin B12 and folate tests are useful for identifying patients with a deficiency. In this issue of the Journal, Willis and colleagues highlight some of the limitations of serum vitamin B12 assays.1 They also emphasise the uncertainty regarding whether red-cell or serum folate should be the preferred first-line test for folate status. The issues underlying some of the data presented require elaboration.

Vitamin B12 assays have an interpretative grey zone in the region of low-normal and mildly low results. Outside the grey zone, the tests show good performance characteristics: a cut-off of 221 pmol/L has a sensitivity of 99%2 and a cut-off of 123 pmol/L has a specificity of 95%.3 Optimal decision points may vary between methods, but results above 220 pmol/L generally rule out deficiency, while results below 125 pmol/L “rule in” deficiency. Between these limits, misclassification may occur if results are interpreted in a binary manner as simply above or below the lower limit of normal (typically about 150 pmol/L). One study used a binary interpretative approach in a cohort of patients with low-normal or low vitamin B12 concentrations (< 221 pmol/L).4 It is the results of this study that have led to the claims of the extraordinary misclassification rate of 50%, quoted by Willis et al. In contrast, the appropriate response to vitamin B12 results of 125–220 pmol/L in patients clinically suspected of deficiency is further testing — using, for example, metabolic markers.

Clinicians also need to be alert to interference in vitamin B12 assays from intrinsic factor antibodies. The interference is sporadic and the same sample may give false results in one assay but not another. An investigation of 23 samples from patients with clinically overt pernicious anaemia (15 positive and eight negative for intrinsic factor antibodies) showed false-normal results for five to eight of the samples, depending on the assay used.5 It is from this report of highly selected samples that claims of “assay failure rates of 22% to 35%” are made.1 This overstates the frequency of the error among non-selected requests to laboratories, which has been estimated as closer to 1 in 3000 requests.6 The interference appears to be limited to samples from overtly deficient patients and therefore needs to be considered when vitamin B12 results are normal or high in patients with clinically evident deficiency.

In most clinical contexts, assessment of folate status is valid with either red-cell or serum folate. Red-cell folate has the theoretical advantage of providing a longer-term assessment of folate status. This is offset in practice by additional variation in red-cell folate measurements due to sample pretreatment factors and the binding of folate to deoxyhaemoglobin — factors that do not influence serum folate results. A systematic review recently assessed the performance of red-cell versus serum folate for identifying deficiency.7 It found that neither was clearly superior, although serum folate more frequently showed higher correlation with homocysteine as a functional marker of deficiency.

Tests for folate status remain relevant among populations in countries where staple foods are fortified with folate. The introduction of mandatory fortification of wheat flour used for breadmaking has reduced the prevalence of deficiency in Australia. In most patients with macrocytic anaemia it may therefore be appropriate to use folate as a second-line investigation after more common causes have been excluded. However, deficiency may still be seen, particularly in those not regularly consuming bread or other grain-based products, such as those with coeliac disease or alcohol dependence.

Vitamin B12 and folate assays are widely available and inexpensive investigations. They identify vitamin deficiencies that have serious consequences if untreated. Recognition of the grey zone in vitamin B12 interpretation limits misclassification; however, clinicians must also remain alert to sporadic assay interference in overtly deficient patients. Folate status may be assessed with either red-cell or serum folate. As the faster and less expensive test to perform, serum folate appears to offer the best combination of test cost and clinical information.