Tests
1st tests to order
CBC
Test
To determine baseline hematocrit, hemoglobin, and MCV.
Useful for diagnosing severe and prolonged vitamin B12 deficiency, but not useful for diagnosing early vitamin B12 deficiency.
Not useful to rule out vitamin B12 deficiency; many patients with documented vitamin B12 deficiency may have normal hematologic parameters.[77]
Result
elevated MCV, low hematocrit
peripheral blood smear
Test
Classic hypersegmented polymorphonucleated cells and megalocytes are seen in severe vitamin B12 deficiency, causing megaloblastic anemia.
Megalocytes are red blood cell precursors whose numbers increase due to the importance of vitamin B12 to hematopoiesis.
Can be normal in early deficiency.
Result
megalocytes, hypersegmented polymorphonucleated cells
serum vitamin B12
Test
Serum vitamin B12 <200 picograms/mL indicates probable vitamin B12 deficiency. Confirmatory diagnosis is generally unnecessary and empiric treatment should begin.[1][2]
Additional testing should be done to rule out possible vitamin B12 deficiency (201-350 picograms/mL).
Patients with serum vitamin B12 >350 picograms/mL are unlikely to have vitamin B12 deficiency. However, vitamin B12 level >350 picograms/mL does not exclude vitamin B12 deficiency, particularly if pernicious anemia is suspected. Spuriously normal or high serum vitamin B12 levels have been reported in patients with pernicious anemia due to anti-intrinsic factor antibody interference in laboratory assays.[3][4][5][6] Further testing for methylmalonic acid, homocysteine, and holotranscobalamin should be carried out to determine if vitamin B12 deficiency is present.
Optimal serum vitamin B12 levels for hematologic and neurologic function are still undetermined.
Result
<200 picograms/mL (probable deficiency); 201-350 picograms/mL (possible deficiency); >350 picograms/mL (unlikely deficiency, but does not rule out a diagnosis, particularly if pernicious anemia is suspected)
reticulocyte count
Test
Used to differentiate B12 deficiency from hemolytic anemia.
Low reticulocyte index indicates decreased production, unlike in hemolytic anemia, in which reticulocyte index would be increased.
Result
low corrected reticulocyte index
Tests to consider
methylmalonic acid (MMA)
Test
A marker of vitamin B12 tissue deficiency.
Caution in renal disease, as elevated MMA levels occur.[1]
Elevated MMA may be spurious and requires subsequent follow-up to determine whether MMA normalizes with adequate treatment.
Result
elevated (>0.4 micromol/L, but may be lab-specific)
homocysteine
Test
A marker of vitamin B12 tissue deficiency.
Not as specific as methylmalonic acid (MMA) for vitamin B12 deficiency.[1] Also elevated in folate deficiency and hypothyroidism.
Result
elevated (>15 micromol/L [>2.03 mg/dL], but may be lab-specific)
holotranscobalamin (hTC)
Test
A marker of vitamin B12 tissue deficiency.
Measures vitamin B12 bound to transcobalamin. Low levels along with low normal serum vitamin B12 suggest inadequate absorption.
Several studies have reported greater diagnostic accuracy with the hTC assay than with other assays measuring markers of vitamin B12 deficiency.[66]
Result
<35 picograms/L is diagnostic
anti-intrinsic factor antibody (anti-IFAB)
Test
Once vitamin B12 deficiency is confirmed, testing for anti-IFAB can determine whether pernicious anemia is the cause. Only 50% sensitive, but highly specific for pernicious anemia.[2]
Testing for anti-IFAB should be done before initiating vitamin B12 replacement therapy because high vitamin B12 levels may lead to false positive results.[70][71]
Result
positive if pernicious anemia is the cause
antiparietal cell (APC) antibody
Test
Once vitamin B12 deficiency is confirmed, APC antibody can, in conjunction with other tests, help to determine whether pernicious anemia (PA) is the cause.
Highly sensitive (85%), but has low specificity for PA because APC antibodies may be elevated in atrophic gastritis.[2]
Result
positive result may suggest PA; positive result is not sufficient for diagnosis of PA because APC antibodies may be elevated in atrophic gastritis
serum gastrin (fasting)
Test
Gastrin levels rise in gastric achlorhydria and can signify pernicious anemia.[72]
Result
elevated if pernicious anemia is the cause
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