Vitamin B12 Deficiency

At a Glance

Diagnosis of vitamin B12 (cobalamin) deficiency is relatively straightforward in long-lasting deficiency, which manifests itself with marked anemia, macrocytosis (mean corpuscular volume [MCV] > 100 fL with ovalocytosis, possibly in conjunction with thrombocytopenia and neutropenia > pancytopenia) neutrophil hypersegmentation (at least 5% of neutrophils with 5 lobes or at least 1% with 6 lobes), and neurologic manifestations (paresthesias and ataxia, with/without mental status changes).

Diagnosis of pernicious anemia is also relatively simple in the case of a patients presenting with fatigue due to anemia, neurologic symptoms (myelo-neuropathic, more rarely cerebral/autonomic), and glossitis. Pernicious anemia is also usually associated with family history of the disease in one-third of the patients and with other autoimmune diseases. However, the majority of cases do not present with this classic, full-blown symptomatology, and, in many cases, the classic manifestations may be absent.

Macrocytosis usually precedes anemia by a few months, but anemia may not be present in up to one-quarter of patients, who may have exclusively neurologic manifestations. Possibly as a result of the folate supplementation mandated by the Food and Drug Administration (FDA) in the United States and the widespread use of multivitamin supplements, purely neurologic presentations seem to prevail, whereas the hematological manifestations seems to have diminished. In addition, much more common is subclinical B12 deficiency, with no clinical expression, but with the presence of abnormal biochemical markers. In these cases, gastrointestinal (GI) absorption is normal in the majority of cases, and small reductions in absorption can be demonstrated for food-bound B12 in one-third to one-half of the cases.

There are several conditions that should raise suspicion for either subclinical or clinically relevant B12 deficiency: Anemia is common among the elderly (older than 65 years of age), and B12 deficiency may contribute to a significant fraction of these cases. Malabsorptive GI diseases, such as sprue, celiac disease (gluten-sensitive enteropathy), partial gastrectomy, short bowel syndrome, inflammatory bowel disease, and bariatric surgery, carry an increased risk of association with B12 deficiency.

Strict vegetarians (veganism, avoidance of all animal products) and chronic alcoholics should also be considered at risk of developing B12 deficiency. Young adults with a history of nitrous oxide (N2O) abuse by inhalation should be considered at risk of developing B12 deficiency. Patients with pancreatic insufficiency are at risk of developing B12 because of the associated malabsorption of B12 that cannot be released from dietary protein and, thus, is unable to bind to intrinsic factor (IF). Infants exclusively fed by breast-feeding or born to strict vegetarian mothers are at increased risk of developing B12 deficiency, which manifests itself primarily with neurologic symptoms and failure to thrive. Patients on chronic therapy with either metformin or omeprazole have been shown at increased risk of developing B12 deficiency. In developing countries, nutritional insufficiencies due to poverty are associated with reduced B12 levels.

In general, since the daily allowance for B12 (2.4 µg/day, to match an estimated daily loss/requirement of 1 µg/day) is quite small compared with the body stores (~2,500 µg), B12 deficiency develops over years in adults. Blood smear morphology and white blood cell (WBC) count: WBC is usually decreased or at the lower limit of normal range.

Hypersegmentation of neutrophils is a useful diagnostic sign, which can be present in conjunction with neurological symptoms and in the absence of anemia. Red cell morphology shows the presence of macrocytes and ovalocytes. Reticulocytes are either normal or reduced.

With the FDA-mandated folic acid supplementation of many grain products, true folate deficiency is now much less common in the United States. However, it is important to keep in mind that folate and B12 deficiency may both be present in the same patient, so, in the work-up of a possible B12 deficiency, serum folate should always be assessed.

What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?

Direct measurement of B12 levels in serum/plasma and of relevant metabolites are required to confirm a diagnostic suspicion of B12 deficiency. In many cases, final confirmation of the diagnosis requires demonstrating normalization of biochemical markers of B12 deficiency following B12 therapy.

Serum/Plasma B12 Levels

Serum values less than 250-200 ng/L (185-148 pmol/L) according to the assay may be indicative of B12 deficiency but, in isolation, are not indicative of the presence of true B12 deficiency, which must be demonstrated by the concordance of clinical and laboratory findings. The sensitivity of a reduced B12 is excellent in a patients with megaloblastic anemias (> 95%) but is dramatically reduced in subclinical B12 deficiencies.

B12 deficiency is highly unlikely when B12 values in plasma are greater than 300 pg/ml (221 pmol/L). Two components are measured with the total serum/plasma B12 assays: one is the so-called active holotranscobalamin (holo-TC II), which determines the supply of B12 to the tissues via a rapid exchange, and the other is the B12 bound to haptocorrin (holohaptocorrin, HoloHC, or Holo-TC I), which is considered metabolically not exchangeable. Only 20-30% of plasma B12 is bound to holo-TC II. Thus, low total B12 levels (lower limit of normal threshold usually ~140 pmol/L) are not automatically associated with tissue B12 deficiency, unless “active” B12 (lower limit of normal ~23 pmol/L) values are also decreased. This is often seen in pregnancy in which the total B12 levels decrease significantly, with most of the changes due to a decrease in the holoHC (inactive) component.

Conversely, an elevated total B12 value may be misleading if due exclusively to an increase in holoHC (inactive), as often seen with increased WBC counts.

Metabolic Intermediates Assays

These assays are useful in cases with equivocal B12 levels and with unexplained neurologic symptoms in the attempt to uncover a cause for dementia other than Alzheimer’s. The serum levels of homocysteine and metylmalonic acid (MMA) are elevated in the presence of B12 deficiency, which also causes elevation of the urinary concentration of MMA. These assays are helpful in the differential diagnosis with folate deficiency, which is associated with increases in serum homocystein, but does not affect MMA metabolism. In overt clinical B12 deficiency, serum values may be greater than 25 microM for homocysteine and 1,000 nmol/L for MMA.

Pernicious Anemia

The Schilling test and the gastrin secretion test have primarily historical value and are not part of the diagnostic work-up for pernicious anemia. Diagnosis is primarily based on the presence of antibody directed against intrinsic factor (IF; the value of antibodies directed against gastric parietal cells being more limited) in conjunction with increased serum levels of gastric and pepsinogen. An increase in serum gastrin and pepsinogen above the normal range is very sensitive for diagnosing pernicious anemia; unfortunately it is not very specific, since these values increase in a variety of conditions other than pernicious anemia. The combined us of antibodies against IF and serum gastrin/pepsinogen partially obviates to the relatively low sensitivity (50-70%) of the antibodies against IF.

Serum Folate

National Health and Nutrition Examination Surveys (NHANES)/Center for Disease Control (CDC) criteria for folate deficiency use a cut-off value for serum folate of 3.0 ng/ml. A value greater than 4 ng/ml (9.1 nmol/L) effectively rules out folate deficiency.

Increased LDH and Bilirubin (mostly direct)

This confirms the presence of ineffective erythropoiesis due to B12 deficiency, which results in altered maturation of erythroid precursors.

Bone Marrow Aspirate and Biopsy

Marrow is hypercellular with characteristic features of megaloblastic erythropoiesis and presence of large metamyelocytes. The bone marrow does not allow us to distinguish B12 from folate deficiency and should be reserved for cases in which there is a suspicion of an underlying hematological malignancy

The presence of intestinal malabsorption of B12 should be carefully investigated. Although testing for antibodies against IF is helpful in identifying pernicious anemia (in conjunction with serum gastrin and pepsinogen), upper endoscopy and small bowel studies should be considered in selected cases. Small-bowel biopsy should be considered in cases with suspected celiac disease in conjunction with anti-endomysial antibodies and anti–tissue-transglutaminase antibodies.

Are There Any Factors That Might Affect the Lab Results? In particular, does your patient take any medications – OTC drugs or Herbals – that might affect the lab results?


Macrocytosis is a common finding in adult patients, with an estimated incidence around 2-3%. The majority of these cases do not have anemia. Causes other than B12 deficiency include folate deficiency, azidothymidine (AZT) therapy, chemotherapy, elevated alcohol intake and/or liver dysfunction, hypothyroidism, myelodysplastic syndrome, and multiple myeloma. Macrocytosis (MCV > 100 fL) may be masked or absent in the presence of concomitant iron deficiency or alpha/beta thal traits, although these cases tend to have an increased red blood cell distribution width (RDW).

Serum B12

Many assays are based on immunological methods, which quantify the competitive binding of B12 by IF. There have been cases of artificially normal B12 values in patients with reduced B12 levels due to the inability of the reagents to denature antibodies against IF in the patient samples, which end up competing with the anti-IF antibody used for the assay. Low B12 values may be due to genetic polymorphisms/defects in the B12-binding proteins.

Transcobalamin I (TC I) is the major carrier of B12 in plasma (80%), and TC I deficiencies are associated with B12 values less than 100 pmol/L when both alleles of this gene are altered. Conversely, elevated TC I levels produce high B12 concentrations in plasma. These are seen in CML, some hepatic tumors, leukocytosis, and renal failure. B12 levels may be falsely elevated and mask true B12 deficiency in liver disease with hepatocellular damage. Falsely low B12 levels have been reported following very high doses of Vitamin C, in multiple myeloma, and in one-third of cases of true folate deficiency.

Serum Folate

Samples should be taken before any meal, since serum folate may transiently normalize following a folate-containing meal. Similar effects have also been described following blood/plasma transfusions. Folate levels can also be below the normal range because of low intake in the preceding few days, during pregnancy, or as a consequence of increased alcohol intake or of therapy with anticonvulsant drug like carbamazepine, phenobarbital, and phenytoin,

Serum/plasma Homocysteine

Values are elevated in the presence of hereditary homocysteinemia.

Serum/Plasma/Urinary Methylmalonic Acid

Antibiotic treatment has been associated in some studies with reduced MMA levels. MMA levels remain elevated during the first year of life. Cutoff values have varied from 210 to 480 nmol/L, with the most commonly used being around 270 nmol/L. Renal failure is associated with increased values of plasma MMA, whereas methylmalonic aciduria increased both serum and urine MMA.

What Lab Results Are Absolutely Confirmatory?

Normalization of serum homocysteine and methyl malonic acid following B12 supplementation is a strong additional confirmation of the diagnosis of B12 deficiency. Incomplete therapeutic response is associated with only transient normalization of these parameters. In many instances, normalization of these parameters provides the most convincing proof of the presence of B12 deficiency.

The reticulocyte count promptly increases after treatment, peaking at 1 week following B12 administration. A blunted response may be indicative of the presence of iron deficiency.

Additional Issues of Clinical Importance

Severe megaloblastic anemia can be misdiagnosed as erythroleukemia. In rare cases, acute myeloid leukemia (AML) can be misdiagnosed as megaloblastic anemia.

Patients with pernicious anemia require regular screening for thyroid disease and iron deficiency. They are also at higher risk of developing gastrointestinal (GI) malignancies, such as carcinoids and gastric cancer.

N2O inactivates B12: complete blood count (CBC) and B12 should be assessed, especially in elderly patients prior to exposure to N2O for dental or minor surgical procedures.

Hypokalemia frequently develops following B12 treatment of severe anemias, but it is rarely of clinical importance/concern.

Iron deficiency may be masked in the presence of severe B12 deficiency. A blunted reticulocyte response following B12 administration is an important sign of this possibility. Further indication may be provided by an incomplete normalization of cellular indices 8 weeks after treatment. Iron studies should be considered, and iron supplements can be given prophylactically in conjunction with B12 administration.

Infections with Helicobacter Pylori have been associated with B12 and folate deficiencies and should be considered in the presence of upper GI symptoms.