Mean corpuscular volume: the multi-purpose screening tool

The mean corpuscular volume (MCV) is used to screen for folate, vitamin B₁₂ and Fe deficiency and is measured as part of the full blood count. Poor folate or vitamin B₁₂ status increases MCV by impairing DNA synthesis and cell division, whereas Fe deficiency decreases MCV as a result of impaired Hb production⁽Reference Greer, Foerster, Lukens, Rodgers, Paraskevas and Glader⁹⁾. The normal range in NPNL adult women is 80–100 fl, although during pregnancy this level is elevated slightly because of the physiological increase in the proportion of younger larger erythrocytes⁽Reference Milman, Bergholt, Byg, Eriksen and Hvas^8, Reference Lurie¹³⁾. When screening for Fe-deficiency anaemia (IDA), MCV is considered alongside Hb and packed cell volume.

The detection of anaemia by MCV tends to occur only after functional deficiency lasting several weeks or more, since it takes time to repopulate the erythrocyte mass with enough cells of abnormal size to affect the mean markedly. Sensitivity is further reduced during pregnancy since the physiological increase in MCV can potentially mask the early stages of microcytosis⁽Reference Milman, Bergholt, Byg, Eriksen and Hvas⁸⁾. It is therefore most useful at the initial booking appointment since it can identify pre-existing micronutrient deficiency that can be treated in time to prevent further deterioration^(14–Reference Nybo, Friis-Hansen, Felding and Milman¹⁶⁾. During late pregnancy, however, it may fail to detect individuals with recently-developed deficiency brought about by the high demand for micronutrients⁽Reference Rusia, Flowers, Madan, Agarwal, Sood and Sikka^17, Reference van den Broek, Letsky, White and Shenkin¹⁸⁾.

Hb: the maternal oxygen-carrying capacity

The conceptus is totally dependent on the maternal supply of O₂ for its own respiration and this supply is delivered to the placenta, bound to Hb, at a rate governed by several factors including cardiac output, ventilation rate, uterine blood flow, smoking, altitude and packed cell volume⁽Reference Carter¹⁹⁾. Insufficient supply of Hb to the placenta caused by maternal anaemia can potentially result in fetal hypoxia with serious adverse consequences for fetal development⁽Reference Mostello, Chalk, Khoury, Mack, Siddiqi and Clark²⁰⁾.

Impaired Hb production during pregnancy is most often caused by poor Fe status, and therefore measurement of Hb serves as a useful screening method for the detection of IDA. Assessment is complicated by the physiological expansion of the plasma volume during early pregnancy and the subsequent increase in erythrocyte mass⁽Reference Lund and Donovan^21, Reference Orsi and Tribe²²⁾. The net result of these haematological changes is a 5–15% dilution of the maternal blood supply, which reduces blood viscosity and improves substrate delivery to the placenta⁽Reference Rasmussen^23, Reference Chesley²⁴⁾. This process is called the ‘physiological anaemia of pregnancy’ and does not reflect any deterioration in maternal Fe status or O₂-carrying capacity.

There is considerable debate as to what constitutes the optimal Hb range during pregnancy. The US Centre for Disease Control has classified maternal anaemia based on data from healthy Fe-supplemented mostly-Scandinavian pregnant women⁽Reference Svanberg, Arvidsson, Norrby, Rybo and Solvell^25–Reference Sjostedt, Manner, Nummi and Ekenved²⁸⁾. Using the 5th percentiles from these groups to define the limit of the normal range, the Centre for Disease Control has defined anaemia as Hb <110 g/l during the 1st and 3rd trimesters and <105 g/l during the 2nd trimester. Similarly-derived cut-off points have also been established for packed cell volumes. A more recent dataset of several hundred healthy pregnant Danish women has found similar results. Normal ranges (g/l), defined as means±1·96 sd, were: 118 (105–132) at 18 weeks of gestation, 118 (103–134) at 32 weeks of gestation, and 124 (108–140) at 39 weeks of gestation⁽Reference Milman, Bergholt, Byg, Eriksen and Hvas⁸⁾. These values are assumed to represent a healthy range of values within these populations, since no clinically-significant adverse pregnancy outcomes were recorded. Hb concentrations below these lower cut-off points are considered likely to reflect maternal IDA to some extent.

The effects of uncomplicated IDA in human pregnancy are difficult to ascertain since ethical considerations often prevent controlled studies from being carried out. One alternative is to observe the relationships between IDA and pregnancy outcomes in free-living populations. Unfortunately, such studies are confounded by factors such as inadequate haemodilution⁽Reference Hytten^29, Reference Salas, Marshall, Gutierrez and Rosso³⁰⁾, smoking⁽Reference Leifert³¹⁾, chronic inflammation⁽Reference van den Broek and Letsky³²⁾, infection⁽Reference Muhangi, Woodburn, Omara, Omoding, Kizito, Mpairwe, Nabulime, Ameke, Morison and Elliott^33, Reference Geelhoed, Agadzi, Visser, Ablordeppey, Asare, O'Rourke, van Leeuwen and van Roosmalen³⁴⁾ and low BMI⁽Reference Geelhoed, Agadzi, Visser, Ablordeppey, Asare, O'Rourke, van Leeuwen and van Roosmalen³⁴⁾, all of which have strong independent effects on pregnancy outcome. Most studies have reported the optimal Hb range in mid-to-late pregnancy to include mild anaemia as defined by Centre for Disease Control values (Hb 95–110 g/l)⁽Reference Chang, O'Brien, Nathanson, Mancini and Witter^35–Reference Steer³⁸⁾ and a few have even questioned the risks associated with moderate or severe IDA (Hb <95 g/l and <80 g/l respectively), since the numerous observational studies that have examined this issue are highly inconsistent⁽Reference Rasmussen²³⁾.

An alternative method is to examine the effects of maternal anaemia in highly-controlled animal models. Most of these studies have used ovine models, although in studies that have been carried out in human subjects using non-invasive techniques the results have generally been in agreement⁽Reference Mostello, Chalk, Khoury, Mack, Siddiqi and Clark^20, Reference Edwards, Novy, Walters and Metcalfe^39, Reference Delpapa, Edelstone, Milley and Balsan⁴⁰⁾. These results show adaptive responses by both mother and fetus to acute falls in maternal packed cell volume that help to prevent maternal and fetal hypoxia. The affinity of Hb for O₂ is reduced by increasing erythrocyte concentrations of 2,3-diphosphoglycerate, which allows more efficient uptake of O₂ by maternal and fetal tissue⁽Reference Edwards, Novy, Walters and Metcalfe^39, Reference Richardson and Bocking⁴¹⁾. Increased maternal cardiac output and utero–placental blood flow maintains the rate of O₂ delivery⁽Reference Mostello, Chalk, Khoury, Mack, Siddiqi and Clark^20, Reference Delpapa, Edelstone, Milley and Balsan⁴⁰⁾ and increased fetal heart rate and blood pressure maintains the distribution of O₂ to fetal tissue⁽Reference Pulgar, Zhang, Massmann and Figueroa⁴²⁾. In addition, both mother and fetus may reduce their rates of physical activity to levels that require less O₂. Only when O₂ delivery falls to below 50% of normal levels, or when conditions persist for several days, does the rate of fetal O₂ consumption fall as a linear function of O₂ delivery⁽Reference Paulone, Edelstone and Shedd^43, Reference Bocking, White, Homan and Richardson⁴⁴⁾. Under such conditions the fetus adapts to the lower supply of O₂ by up regulating erythropoiesis and down regulating fetal growth, both of which help to prevent the onset of hypoxia or acidosis in all but the most extreme conditions.

Whilst the effects of mild maternal anaemia remain highly controversial, moderate-to-severe anaemia in both animal models and human observational studies has been consistently associated with fetal growth restriction, probably mediated by chronically-impaired maternal O₂-carrying capacity⁽Reference Mostello, Chalk, Khoury, Mack, Siddiqi and Clark^20, Reference Richardson and Bocking^41, Reference Mahajan, Aalinkeel, Shah, Singh and Kochupillai^45, Reference Spinillo, Capuzzo, Piazzi, Nicola, Colonna and Iasci⁴⁶⁾. However, it remains unclear whether there is any discernable benefit of Fe therapy in terms of pregnancy outcomes, other than restoration of maternal Fe status⁽Reference Reveiz, Gyte and Cuervo¹⁵⁾. The continuing practice of prophylactic Fe supplementation is based on the general consensus that Fe deficiency is by definition undesirable and that its prevention is more likely to be beneficial than harmful⁽⁴⁷⁾. Maternal supplementation during pregnancy is also likely to improve neonatal Fe status and thus protect the infant from IDA, especially if born preterm^(47–Reference Singla, Gupta and Agarwal⁴⁹⁾.

Erythrocyte zinc protoporphyrin

The most established biomarker of IDE is zinc protoporphyrin (ZnPP), which measures the extent to which Zn, rather than Fe, has been chelated with protoporphyrin. Increasing amounts of ZnPP are produced in maturing erythroblasts as Fe availability becomes suboptimal in the bone marrow. This process may function as a homeostatic mechanism by inhibiting the excretion of Fe following haemolysis by macrophages⁽Reference Maines⁷⁸⁾. Thus, it is a highly-sensitive functional indicator of IDE, especially when presented as ZnPP:haem, as this variable controls for haemodilution during pregnancy⁽Reference Schifman, Thomasson and Evers⁷⁹⁾. ZnPP remains constant throughout pregnancy in Fe-replete women, whereas in unsupplemented women it tends to rise markedly in the last trimester⁽Reference Romslo, Haram, Sagen and Augensen^80, Reference Milman, Ibsen and Christensen⁸¹⁾.

Although ZnPP measures IDE with reasonable accuracy, it cannot distinguish between true and functional Fe deficiency. The latter occurs when body Fe stores are adequate yet Fe is not available to the bone marrow, such as can happen during infection and inflammation. The underlying cause of IDE may be identified by concurrent assessment of serum ferritin. For instance, if both measures are high then IDE caused by chronic inflammation is more likely, whereas if ZnPP is high (≥60 μmol/mol haem) and serum ferritin is low (<15 μg/l) then true IDE caused by depleted Fe stores is more likely. This combined use of ZnPP and ferritin values has been recommended by several authors as a reliable method for the determination of IDE⁽Reference Hastka, Lasserre, Schwarzbeck and Hehlmann^82, Reference Labbe, Vreman and Stevenson⁸³⁾. However, it should be noted that functional and true IDE are not mutually exclusive processes and may co-exist, especially during late pregnancy when low-level inflammation with depleted Fe stores is a common occurrence. In such circumstances the relative contributions of the underlying causes of IDE may be difficult to quantify.

The main advantages of ZnPP are its cost efficiency and its ability to be used in the field by unspecialised personnel using a portable haematofluorometer. However, its use is limited by its high sensitivity to environmental Pb pollution⁽Reference Lamola and Yamane⁸⁴⁾, which inhibits the binding of Fe to protoporphyrin, and its poor sensitivity in identifying improvements once Fe therapy has begun. This limitation might be resolved if the assay could be refined to measure only reticulocyte ZnPP, as has been suggested by some authors, and initial crude efforts to this effect have shown considerable promise⁽Reference Kleven, Blohowiak and Kling⁸⁵⁾. Despite its advantages, however, the lack of an automated method for assessment of ZnPP has severely constrained its use in most clinical laboratories.

Reticulocyte indices

Reticulocytes are immature erythrocyte cells that under normal conditions comprise about 1% of the erythrocyte population. They mature in the bone marrow for 1–3 d and are then released in the maternal circulation for 1–2 d before they lose their RNA and become fully mature. Reticulocyte counts have been available in most clinical laboratories for many years as a measure of the rate of erythropoiesis. However, a few modern haematological analysers now have the ability to measure the Hb content of reticulocytes, which effectively measures the extent of IDE over the previous 3–4 d⁽Reference Brugnara⁸⁶⁾.

Reticulocyte Hb content (CHr) is similar in some aspects to ZnPP. It is a cellular measure of IDE that on its own does not distinguish between true and functional Fe deficiency. However, unlike ZnPP, analysis is restricted to reticulocytes, so its sensitivity to short-term changes in Hb production is much greater⁽Reference Thomas and Thomas⁸⁷⁾. Values <26–28 pg identify IDE in NPNL adults⁽Reference Fishbane, Galgano, Langley, Canfield and Maesaka⁸⁸⁾.

CHr works by assessment of the reticulocyte cell volume and Hb content; hence conditions that change MCV independently of Fe deficiency will lower the precision of Chr⁽Reference Mast, Blinder and Dietzen⁸⁹⁾. Such conditions include microcytosis caused by thalassaemia and macrocytosis caused by folate or vitamin B₁₂ deficiency⁽Reference Mast, Blinder, Lu, Flax and Dietzen⁹⁰⁾. Unfortunately, concurrent Fe and folate deficiency is relatively common during late pregnancy⁽Reference d'Onofrio, Chirillo, Zini, Caenaro, Tommasi and Micciulli⁹¹⁾. Whilst initial validation studies seem to suggest that CHr is an accurate method of assessing IDE in pregnancy, the effects of suboptimal folate or vitamin B₁₂ status may be important sources of error during pregnancy that require further study.

CHr is essentially a short-term indicator, but it can now be measured alongside the percentage of hypochromic erythrocytes, which until recently had only been available by manual blood film⁽Reference Brugnara⁸⁶⁾. Together CHr and the percentage of hypochromic erythrocytes appear to offer the most accurate assessment of past and present IDE and IDA during pregnancy⁽Reference Ervasti, Kotisaari, Heinonen and Punnonen⁹²⁾. However, determination of the underlying cause of IDE may require additional assessment of tissue Fe levels.

Serum soluble transferrin receptor

Soluble transferrin receptors (sTfR) are truncated versions of the transferrin receptors that facilitate cellular uptake of Fe-rich plasma transferrin. Expression of transferrin receptors is inversely related to that of intracellular ferritin, since both are mediated by Fe-responsive elements whose main purpose is to maintain intracellular Fe concentrations⁽Reference Klausner, Rouault and Harford¹⁰⁷⁾. Expression of transferrin receptors therefore increases as tissue Fe levels fall, resulting in higher concentrations of sTfR in the serum⁽Reference Skikne, Flowers and Cook¹⁰⁸⁾. The density of transferrin receptors is highest in cells with the greatest demand for Fe, such as immature erythroid cells, hepatocytes and the placenta⁽Reference Huebers and Finch¹⁰⁹⁾.

Table 1. Laboratory measures of maternal iron status

sTfR, soluble transferritin receptor; ZnPP zinc protoporphyrin; CHr, reticulocyte Hb content; MCV, mean corpuscular volume; Hct, packed cell volume; percentage of hypochromic erythrocytes; ↑, increased; ↓, reduced.

Serum sTfR has a high specificity to tissue Fe deficiency and a low sensitivity to acute or chronic inflammation, unlike most other indicators of Fe status⁽Reference Ferguson, Skikne, Simpson, Baynes and Cook^110, Reference Dimitriou, Stiakaki, Markaki, Bolonaki, Giannakopoulou and Kalmanti¹¹¹⁾. When combined with measurement of serum ferritin, the sTfR:ferritin can be used to estimate total body Fe (mg/kg) with high precision⁽Reference Cook, Flowers and Skikne¹¹²⁾.

However, the accuracy of sTfR and sTfR:ferritin during pregnancy has not yet been firmly established. As a large proportion of sTfR is derived from eythroid precursors in the bone marrow, changes in the rate of erythropoiesis also affect sTfR concentrations. In Fe-replete individuals sTfR is an accurate indicator of erythropoiesis⁽Reference Robach, Fulla, Westerterp and Richalet¹¹³⁾, whereas in those individuals with a steady rate of erythropoiesis sTfR is an accurate indicator of tissue Fe depletion⁽Reference Metzgeroth, Adelberger, Dorn-Beineke, Kuhn, Schatz, Maywald, Bertsch, Wisser, Hehlmann and Hastka¹¹⁴⁾.

Unfortunately, pregnancy is associated with marked changes in both erythropoiesis and body Fe stores that introduce considerable uncertainty as to the primary cause of changes in sTfR concentrations. There are few data on the normal patterns of erythropoiesis during pregnancy, mainly because of the prohibitive nature of radio Fe studies, and therefore it is not possible to adjust for the influence of erythropoiesis at each stage. During pregnancy raised sTfR may in fact be more strongly predicted by the rate of erythropoiesis than by Fe status⁽Reference Choi, Im and Pai¹¹⁵⁾.

Some studies have reported sTfR to be a sensitive and specific measure of tissue Fe deficiency in pregnancy⁽Reference Akesson, Bjellerup, Berglund, Bremme and Vahter^116, Reference Carriaga, Skikne, Finley, Cutler and Cook¹¹⁷⁾, but these have not had the benefit of comparison with gold standard methods, as has been the case for NPNL subjects. sTfR and the sTfR:ferritin appear to have great potential as indicators of tissue Fe depletion, but their validity during pregnancy remains uncertain until the effects of erythropoiesis can be ascertained. In the meantime, sTfR:ferritin is probably of most use as a method of evaluating the effectiveness of interventions to improve Fe status during pregnancy, since the error as a result of erythropoiesis and inflammation ought to be similar in both control and intervention groups⁽Reference Cook, Flowers and Skikne¹¹²⁾.

Serum total homocysteine

Functional indicators can provide useful information that direct measurement of vitamin concentrations cannot, and are often more sensitive. A widely-used functional biomarker of mild or occult folate depletion is serum total homocysteine (tHcy). tHcy is primarily a functional marker of folate status in Western populations, but is also affected by the status of other nutrients such as vitamin B_12, vitamin B_6, methionine, betaine, choline and riboflavin, all of which are involved in homocysteine production or excretion⁽Reference McNulty, Dowey, Strain, Dunne, Ward, Molloy, McAnena, Hughes, Hannon-Fletcher and Scott^141–Reference Refsum, Nurk, Smith, Ueland, Gjesdal, Bjelland, Tverdal, Tell, Nygård and Vollset¹⁴³⁾.

A major advantage of tHcy over serum folate is that it does not require fasting blood samples, the collection of which may be ethically prohibited in studies of pregnant women. Postprandial serum tHcy concentrations are generally slightly lower than fasted values but these differences are small compared with the total amount of day-to-day variation⁽Reference Thirup and Ekelund^144, Reference Jacques, Rosenberg, Rogers, Selhub, Bowman, Gunter, Wright and Johnson¹⁴⁵⁾. Serum folate, on the other hand, can reflect recent intake, especially when consumed in relatively high doses such as those found in antenatal folic acid supplements⁽Reference Pentieva, McNulty and Reichert¹⁴⁶⁾.

During pregnancy the correlation between folate status and tHcy becomes weaker with advancing gestation. Serum tHcy falls markedly during the first 20 weeks of gestation to approximately half that of NPNL levels and then increases slowly back to pre-conceptual levels by parturition⁽Reference Cikot, Steegers-Theunissen, Thomas, de Boo, Merkus and Steegers¹⁴⁷⁾. This uncoupling with folate concentrations, which in unsupplemented subjects continue to fall throughout pregnancy, remains poorly understood. The decline in serum tHcy in the first half of pregnancy has been suggested to be the result of plasma volume expansion, increased renal clearance, decreased serum albumin and higher concentrations of oestrogens, most of which have been eliminated as causal factors⁽Reference Bartha, Romero-Carmona and Comino-Delgado⁷³⁾. Oestrogens have been suggested as the most probable mediators, but their homocysteine-lowering effects have recently been questioned⁽Reference Murphy, Scott, McPartlin and Fernandez-Ballart^148, Reference Roopnarinesingh, Jackson, Osman, Harrison and Mayne¹⁴⁹⁾.

Despite its idiosyncratic behaviour during pregnancy, serum tHcy remains a sensitive functional biomarker of folate status in pregnant women. Precision may be further increased by adjustment for confounding factors such as renal function, smoking, vitamin B₁₂ status and coffee intake⁽Reference Refsum, Smith and Ueland^150, Reference Grubben, Boers, Blom, Broekhuizen, de Jong, van Rijt, de Ruijter, Swinkels, Nagengast and Katan¹⁵¹⁾. A plasma tHcy concentration >15 μmol/l is generally used to define mild hyperhomocysteinaemia in NPNL women, but because of the physiological decrease in plasma tHcy concentrations during pregnancy, an upper reference limit of 10 μmol/l has been recommended by an expert panel⁽Reference Refsum, Smith and Ueland¹⁵⁰⁾. Whilst there have been problems with tHcy assessment in the past, most modern assays now have good levels of precision and comparability⁽Reference Refsum, Smith and Ueland¹⁵⁰⁾.

p-Amino- and acetamido-benzoylglutamate

The rate of folate turnover can be quantified by measurement of the folate catabolites p-amino-benzoylglutamate and acetamido-benzoylglutamate present in serum or urine⁽Reference McPartlin, Courtney, McNulty, Weir and Scott¹⁵²⁾. Until recently, the analysis procedure has required extensive sample preparation and clean up⁽Reference McNulty, McPartlin, Weir and Scott¹⁵³⁾, making it unsuitable for clinical or epidemiological use. However, recent innovations using liquid chromatography–MS–MS have simplified this process greatly, such that high-volume analysis of urinary and serum p-amino-benzoylglutamate and acetamido-benzoylglutamate may be both possible and cost effective in the future⁽Reference Sokoro, Etter, Lepage, Weist, Eichhorst and Lehotay^154, Reference Hannisdal, Svardal and Ueland¹⁵⁵⁾. Analysis of p-amino-benzoylglutamate may also have potential as a biomarker of folate status in degraded blood samples⁽Reference Hannisdal, Svardal and Ueland¹⁵⁵⁾.

Measurement of folate turnover is not a measurement of folate status per se. It does, however, provide detailed information on an individual's folate requirement, since the amount excreted can be assumed to be the amount required to replace it in order to prevent depletion. The analysis of p-amino-benzoylglutamate and acetamido-benzoylglutamate, together with conventional assessment of folate status, may enable more detailed examination of the relationships between folate demands over the course of pregnancy and maternal folate status, which hitherto has been restricted to samples of less than ten individuals⁽Reference McPartlin, Courtney, McNulty, Weir and Scott^152, Reference Gregory, Caudill, Opalko and Bailey¹⁵⁶⁾.

Serum cobalamin

The accurate detection of vitamin B₁₂ deficiency during pregnancy is extremely difficult. In normal NPNL subjects and in patients with pernicious anaemia serum cobalamin generally correlates well with tissue concentrations, although kinetic studies have shown that concentrations in serum tend to be preserved at the expense of those in tissue⁽Reference Adams, Tankel and MacEwan^165, Reference Boddy and Adams¹⁶⁶⁾. As a result, low serum cobalamin is only likely to become apparent with moderate or severe tissue deficiency. Serum cobalamin of <150 ng/l is generally associated with clinically-apparent deficiency in the NPNL state, yet the usefulness of this, or indeed any, cut-off during pregnancy is questionable⁽Reference Metz, McGrath, Bennett, Hyland and Bottiglieri¹⁶⁷⁾. Concentrations below this level have been found in pregnant women with dietary intakes well in excess of the US RDA of 2·6 μg/d and no corroborative clinical or biochemical evidence of deficiency⁽Reference Pardo, Peled, Bar, Hod, Sela, Rafael and Orvieto^168, Reference Koebnick, Heins, Dagnelie, Wickramasinghe, Ratnayaka, Hothorn, Pfahlberc, Hoffmann, Lindemans and Leitzmann¹⁶⁹⁾.

Functional biomarkers: serum total homocysteine and serum methylmalonic acid

As serum cobalamin is less sensitive during pregnancy, mild or occult vitamin B₁₂ deficiency may be more easily detected with functional biomarkers such as serum methylmalonic acid and serum tHcy. Vitamin B₁₂ has only two functions in animal cells and impairment of either causes the accumulation of unmetabolised precursors. Methylmalonic acid becomes elevated as a result of the impairment of the cobalamin-dependent enzyme methylmalonyl-CoA mutase, which converts methylmalonyl-CoA to succinyl-CoA, an essential precursor of haem and an intermediate in the citric acid cycle. Serum tHcy accumulates as a result of the impairment of cobalamin-dependent methionine synthase⁽Reference Allen, Stabler, Savage and Lindenbaum¹⁷⁰⁾.

Although methylmalonic acid and tHcy are more sensitive than serum total cobalamins, both have major limitations during pregnancy. Serum tHcy can be elevated by folate deficiency, which is more common than vitamin B₁₂ deficiency in Western countries, and becomes uncoupled from vitamin B₁₂ status with advancing gestation. Methylmalonic acid is also affected by renal clearance as well as concurrent use of antibiotics⁽Reference Lindenbaum, Savage, Stabler and Allen¹⁷¹⁾, and can sometimes be sporadically elevated during pregnancy for reasons as yet unknown⁽Reference Metz, McGrath, Bennett, Hyland and Bottiglieri^167, Reference Pardo, Peled, Bar, Hod, Sela, Rafael and Orvieto¹⁶⁸⁾. Measurement of methylmalonic acid is also expensive and requires the use of techniques that may not be widely available in clinical laboratories. The use of either biomarker alone is therefore insufficient to determine vitamin B₁₂ status during pregnancy, although they can provide useful supplemental data to other more direct measures.

Holo-transcobalamin

Cobalamins in human serum are transported by the two main binding proteins, haptocorrin and transcobalamin (TC). These proteins are either complexed with cobalamins (holo) or cobalamin-free (apo). Haptocorrin carries most of the cobalamins in serum, which are cleared slowly over a period of several days. However, no major function has yet been attributed to it other than the binding of metabolically-inert forms of vitamin B₁₂. TC, on the other hand, is highly active and serves as the main transporter of cobalamins from the intestine to tissues including the placenta, which is rich in TC receptors⁽Reference Obeid, Morkbak, Munz, Nexo and Herrmann¹⁷²⁾. Newly-absorbed cobalamins bound to holo-TC are generally cleared from the maternal circulation within minutes, although a small amount remains in circulation.

Recent longitudinal analysis of TC and haptocorrin fractions during pregnancy has determined the reason for the decline in serum cobalamin with advancing gestation⁽Reference Morkbak, Hvas, Milman and Nexo⁷⁾. Large reductions were found in the haptocorrin fraction saturated with true cobalamins, whereas holo-TC remained stable over the whole of gestation. This finding explains why the physiological reduction in serum cobalamin during pregnancy is not associated with functional vitamin B₁₂ deficiency, as haptocorrin is relatively inert.

Since TC is the main fraction available for placental uptake of vitamin B₁₂, maternal serum holo-TC concentrations are highly correlated with cord blood cobalamins and therefore reflect the maternal supply of vitamin B₁₂ to the fetus⁽Reference Obeid, Morkbak, Munz, Nexo and Herrmann¹⁷²⁾. Whilst the high sensitivity and specificity of maternal holo-TC makes it the most suitable biomarker for the assessment of maternal vitamin B₁₂ status, the assay required for its assessment is still highly-specialised and not yet widely available. Until such time as it is suitable for high-volume low-cost throughput of samples, routine screening for maternal vitamin B₁₂ deficiency will continue to be carried out using crude indicators with low sensitivity and specificity such as MCV and serum cobalamin, leaving many pregnant women at risk of subclinical vitamin B₁₂ deficiency.