Laboratory Studies
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Clinical evidence of vitamin B-12 deficiency
Serum cobalamin levels are the initial test.
Two assays exist: radioassay and the nonradioisotopic assay, chemiluminescence, which is becoming more popular because of improved automation, safety, and cost. Chemiluminescence has a higher reference range value, from 250-1100 pg/mL versus 170-900 pg/mL for radioassay. Using the radioassay and elevated homocysteine (HC) and methylmalonic acid (MMA) as criterion standards, levels are less than 200 pg/mL in 90-95% of patients, 200-300 pg/mL in 5-10%, and greater than 300 pg/mL in 0.1-1%. Be aware of the assay used and how the reference range was determined. A serum cobalamin level that is within the reference range does not exclude cobalamin deficiency.
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Abnormally low vitamin B-12 levels: Test for PA by measuring antibodies against IF.
Antiparietal cell antibodies are present in 90% of PA cases. In patients older than 70 years, 10% have false-positive abnormal antibody levels.
IF antibodies are present in 60% of patients. These are more specific but less sensitive.
If either antibody is positive, the diagnosis of PA is confirmed and further testing is not required.
If antibodies are negative, obtain a serum gastrin level to test for achlorhydria, which is associated with PA. If these are elevated, the diagnosis is likely PA. If these results are normal, perform a Schilling test.
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Borderline vitamin B-12 level and clinical features of vitamin B-12 deficiency: Measure methylmalonic acid (MMA) and homocysteine (HC).
Both folate and vitamin B-12 deficiency can lead to metabolite elevation.
In vitamin B-12 deficiency, MMA and HC are elevated, although HC elevation occurs by itself. MMA is more sensitive than HC.
In folate deficiency, MMA is within the reference range and HC is elevated.
MMA and HC are considered abnormal when greater than 3 standard deviations above the mean. Reference range values are not age dependent for MMA and are 70-350 nM/L. For patients younger than 60 years, reference range values are 5-15 µM/L for HC. In people older than 60 years, the cutoff for HC is 20 µM/L.
If both metabolites are within the reference range, vitamin B-12 deficiency is effectively ruled out. Only 0.2% of 400 patients with low serum vitamin B-12 had normal metabolite levels, and 10% of 98 patients with folate deficiency had metabolite levels within the reference range. False-positive elevations in MMA and HC occur in inborn errors of metabolism, renal disease, and deficiencies of folate. If either metabolite is elevated, test for PA or use the Schilling test.
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Schilling test: The Schilling test is used to determine the etiology of vitamin B-12 deficiency in patients with normal IF antibodies.
Stage 1: Administer radiolabeled cobalamin 0.5-2.0 mCi PO to fasted patients. One to 6 hours later, administer unlabeled cobalamin 1000 mcg IM to saturate transcobalamin and flush hepatic storage. Measure the percentage of radiolabeled cobalamin in a 24-hour urine specimen. Urinary excretion within the reference range is 10-35% over 24 hours. Reduced urinary excretion of cobalamin, ie, less than 7-9% based on individual laboratory reference range values, in persons with normal renal function supports decreased absorption of oral cobalamin. If excretion is low, proceed to stage 2.
Stage 2: Stage 1 is repeated with coadministration of porcine IF 60 mg. If the absorption of cobalamin is normalized, the presumptive diagnosis is PA. If poor absorption persists after administration, proceed to stage 3.
Stage 3: Tetracycline is administered for 5 days prior to reperformance of stage 1 to exclude blind loop as the etiology.
Stage 4: Pancreatic enzymes are administered with repetition of stage 1 to test for pancreatic disease.
Caveats: If vitamin B-12 is administered 48 hours before the Schilling test, dilution of the radiolabeled cobalamin and spuriously low apparent urinary excretion and false-positive results occur. False-negative values occur in food-bound malabsorption due to achlorhydria. True negative results are from dietary deficiencies (vegan) and cobalamin binding–protein abnormalities.
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Routine hematologic and chemistry tests
Hematologic abnormalities may be absent at the time of neurologic presentation.
Vitamin B-12 deficiency produces the classic picture of macrocytic anemia, with a mean corpuscular value (MCV) greater than 100 fL. The MCV correlates with estimated vitamin B-12 level:
MCV of 80-100 fL (normal) indicates less than 25% probability of vitamin B-12 deficiency.
MCV of 115-129 fL indicates a 50% probability.
MCV greater than 130 fL indicates a 100% probability.
Peripheral blood smear shows macro-ovalocytosis, anisocytosis, and poikilocytosis, as well as basophilic stippling of the erythrocytes and Howell-Jolly bodies. Reticulocyte count can be within the reference range or low. Hypersegmentation (>5% of neutrophils with > 5 lobes or 1% with > 6 lobes) of polymorphonuclear cells may occur without anemia. Thrombocytopenia is observed in approximately 50% of patients, and platelets often have bizarre size and shape.
Serum indirect bilirubin and lactate dehydrogenase (LDH) may be elevated because PA can have a hemolytic component.
Achlorhydria is present in many patients with PA.
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Laboratory parameters after administration of vitamin B-12
Anemic patients
Reticulocytosis starts in 3-4 days and peaks at 1 week.
Hemoglobin concentration rises in 10 days and returns to the reference range in 8 weeks.
LDH falls within 2 days.
Hypersegmented neutrophils disappear in 1-2 weeks.
Patients with severe anemia and borderline-to-low iron stores
Serum iron level falls within 24 hours because of increased erythropoiesis.
Hypokalemia may develop because of increased potassium utilization in hematopoiesis.
Imaging Studies
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Because of the increased incidence of gastric cancer in PA, gastric radiographic series are suggested at the first visit.
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In patients with myelopathy, MRI may reveal regional T2 and fluid-attenuated inversion recovery (FLAIR) hyperintensities mainly in the thoracic posterior columns with possible extension into the brain stem. In patients with chronic disease, atrophy of the spinal cord is observed.
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Brain MRI may show T2 and FLAIR hyperintensities in the cerebral white matter and around the fourth ventricle.
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Brain MRI of infants with vitamin B-12 deficiency may show delayed myelination.
Other Tests
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Abnormal evoked potentials may be the first electrodiagnostic finding, even in asymptomatic patients with normal neurologic examination findings. The abnormalities are often referable to a central conduction defect; however, peripheral nerves are also affected.
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Somatosensory evoked potentials (SSEP) may reveal prolongation of L3-P27 latency, reflecting a defect in conduction in the large-fiber sensory pathway between the cauda equina and the contralateral sensory cortex.
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Visual evoked potential (VEP) findings are as follows:
VEP findings may be abnormal even without visual symptoms or signs.
Prolongation of the P100 waveform can be unilateral or bilateral.
P100 may normalize after cobalamin replacement.
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Nerve conduction study (NCS)/EMG findings are as follows:
In 1943, Dynes and Norcross found evidence of neuropathy in 23% of patients with PA. [21]
In a recent study, up to 65% of untreated patients had peripheral neuropathy.
Axonal sensorimotor polyneuropathy is present in up to 80%. Demyelinating or mixed forms are less frequent.
Typical features are decreased conduction velocities and motor or sensory amplitude and denervation on EMG.
In 1991, Healton et al found decreased motor nerve conduction velocities (NCV), absent or reduced sensory potentials, and fibrillations in the distal muscles indicating mixed demyelinating-axonal disease in 7 of 9 cases of vitamin B-12 deficiency and neuropathy. [19]
In 1998, Steiner et al described 5 patients with demyelinating polyneuropathy. [22]
Sensory nerves are usually more affected than motor nerves and are more severely affected distally than proximally. Proximal focal conduction block has been reported, which reversed on treatment.
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Electroencephalography (EEG) findings may be normal or show nonspecific slowing. Follow-up EEG findings may be improved in response to treatment.
Procedures
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Bone marrow aspiration may be performed for histologic examination.
Histologic Findings
The CNS is better characterized than the PNS in vitamin B-12 deficiency. The classic picture is subacute combined degeneration of the spinal cord involving the dorsal columns and corticospinal tracts. Lesions are concentrated in the cervical and upper thoracic cord and the cerebrum.
Spinal cord findings
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Macroscopic: It may be shrunken in anteroposterior diameter. The posterior and lateral columns may be gray-white in color.
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Microscopic: Multifocal vacuolated and demyelinated lesions exist in the white matter of the posterior and lateral columns. Early in the course, myelin sheaths are swollen, but axons appear normal; the largest fibers are predominantly affected. Demyelination starts in the center of the posterior columns of the upper thoracic cord. Then, lesions spread laterally and cranially to the lateral corticospinal tracts in the cervical segments and the medulla. Myelin breakdown, foamy macrophages, and occasional lymphocytes are characteristic, predominantly in a perivascular location. As demyelination and vacuolation increase, axons degenerate. Gliosis may be present in older lesions.
Electronic microscopic findings have only been used in nonhuman primates, not in humans. In the rhesus monkey model, the posterior and lateral spinal cord shows myelin degeneration characterized by separation of myelin lamellae and formation of intramyelin vacuoles leading to complete destruction of the myelin sheath and later to degeneration and loss of axons and gliosis.
Vitamin B-12 deficiency myelopathy is virtually indistinguishable from vacuolar myelopathy in AIDS, which is also characterized by vacuolation between the myelin sheaths accompanied by macrophage infiltration. However, the presence of multinucleated giant cells is characteristic of AIDS and not of vitamin B-12 deficiency.
Brain findings
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Macroscopic: The brain appears normal.
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Microscopic: Findings resemble the spinal cord pathology with scanty small perivascular foci of demyelination within the white matter featuring myelin swelling and axon degeneration. The optic nerve typically shows degeneration, predominantly in the papillomacular bundles.
Peripheral nervous system findings
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Unlike EMG/NCS findings, which indicate predominantly axonal or mixed axonal-demyelinating neuropathy, histologic examination suggests either primary demyelinating or axonal neuropathy with secondary demyelination. However, most studies were performed before the introduction of semithin, teased fiber, and electron microscopy analysis.
A biopsy of the anterior tibial nerve recorded loss of myelin sheath and no loss of axons. [23]
A sural biopsy revealed loss of myelinated fibers and no demyelination in teased fibers, indicating secondary demyelination. [24]
In 1959, Coers and Woolf found endplate and preterminal axon abnormalities in muscle biopsies of patients with cobalamin deficiency, suggesting axonal damage. [25]
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The pathology is better described in animal models. In cobalamin-deficient rats, electron microscopy revealed intramyelin and endoneural edema, with no or minimal axonal damage (reversible on administration of cobalamin) in nerves, dorsal root ganglia, and the ventral and dorsal roots.
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Dalla Torre et al report 2 patients with isolated sensory axonal neuropathy secondary to vitamin B12 deficiency who recovered after cyanocobalamin replacement. [26]
Nonneurologic findings
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Bone marrow hyperplasia
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Mild-to-moderate splenomegaly
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Increased iron deposits in the reticuloendothelial system
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Atrophy in all layers of the stomach, sparing the pyloroduodenal region
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Vitamin B-12–associated neurological diseases. Cobalamin and folate metabolism. TS = thymidylate synthase, DHFR = dihydrofolate reductase, SHMT = serine methyl-transferase.
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Vitamin B-12–associated neurological diseases. Cobalamin deficiency leads to reduced adenosylcobalamin, which is required for production of succinyl-CoA. D-methylmalonyl-CoA is converted to methylmalonic acid.
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Vitamin B-12–associated neurological diseases. Pernicious anemia. Characteristic lemon-yellow pallor with raw beef tongue lacking filiform papillae. Photo from Forbes and Jackson with permission.
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Vitamin B-12–associated neurological diseases. Fluid attenuated inversion recovery (Flair) MRI sequence in a patient with cobalamin deficiency and neuropsychiatric manifestations. Discrete areas of hyperintensities are present in the corona radiata.
