Clinical manifestations and diagnosis of vitamin B12 and folate deficiency

INTRODUCTION — This topic review discusses the symptoms, clinical manifestations, and diagnosis of vitamin B12 and folate deficiency, vitamins that are required for normal hematopoiesis and neurologic function. Vitamin B12 and folate deficiencies are often considered together, although folate deficiency has become less common in individuals who are living in developed countries and consuming a normal diet.

Separate topic reviews discuss the treatment of these deficiencies, their causes and pathophysiology, and other causes of macrocytic anemia:

●Treatment – (See "Treatment of vitamin B12 and folate deficiencies".)

●Pathophysiology – (See "Causes and pathophysiology of vitamin B12 and folate deficiencies".)

●Other macrocytic anemias – (See "Macrocytosis/Macrocytic anemia".)

TERMINOLOGY — Vitamin B12 and folate are both water-soluble B vitamins required for formation of hematopoietic cells (red blood cells, white blood cells, and platelets) (table 1). (See "Overview of water-soluble vitamins".)

●Vitamin B12 – Vitamin B12 is also called cobalamin (Cbl). It is present in foods derived from animal products. (See "Causes and pathophysiology of vitamin B12 and folate deficiencies", section on 'Dietary sources and RDI'.)

The endogenous forms include cobalamin and holotranscobalamin, which represent the active fraction of plasma cobalamin. The supplemental forms used to treat vitamin B12 deficiency include cyanocobalamin, which contains a cyanide (CN) group introduced during chemical synthesis, and hydroxocobalamin.

Cyanocobalamin is predominantly used in the United States, while hydroxocobalamin is predominantly used in Europe; both are effective in treating vitamin B12 deficiency. The major difference between the two preparations is in the pharmacokinetics and dosing interval, as discussed separately. (See "Treatment of vitamin B12 and folate deficiencies", section on 'Available therapeutic preparations'.)

●Pernicious anemia – Pernicious anemia (PA) refers to vitamin B12 deficiency caused by autoantibodies that interfere with vitamin B12 absorption by targeting intrinsic factor (IF), gastric parietal cells, or both. At the time it was described, PA was associated with continuous worsening of symptoms and even death without an available treatment.

●Folate – Folate is also called vitamin B9. The terms "folate" and "folic acid" are sometimes used interchangeably. Technically, the vitamin is found in nature as a folate, while folic acid is the synthetic form used therapeutically; it is an oxidized, water-soluble form that does not exist in nature [1]. Dietary folates are also called folate polyglutamates [2]. Dietary folate is found in plant-based foods and fortified grains. (See "Causes and pathophysiology of vitamin B12 and folate deficiencies", section on 'Dietary sources and RDI'.)

Folinic acid (leucovorin, N5-formyl-tetrahydofolate [THF], 5-formylTHF) and 5-methyltetrahydrofolate (5-MTHF) are naturally occurring forms of reduced folate [1]. The roles of vitamin B12 and folates in this pathway are illustrated in the figure (figure 1). Folinic acid is typically used to prevent toxicities of methotrexate and to potentiate cytotoxicity of fluorouracil (FU) in chemotherapy regimens for colon cancer. This is because folinic acid is rapidly converted to the metabolically active form of folate required in cells (tetrahydrofolate) without the need for dihydrofolate reductase, which is inhibited by methotrexate. Folic acid, folinic acid, and 5-MTHF are all effective in treating folate deficiency.

●Homocysteine and methylmalonic acid – Homocysteine is methylated to create methionine in a reaction that requires folate and vitamin B12 (folate is a methyl donor) (figure 2). This is why both folate and vitamin B12 deficiencies result in accumulation of homocysteine (figure 3).

Methylmalonic acid (MMA) is converted to succinyl-CoA in a reaction that requires vitamin B12 but not folate. This is why vitamin B12 deficiency causes accumulation of MMA but folate deficiency does not.

Use of metabolite testing in diagnosis is discussed below. (See 'Metabolite testing (MMA and homocysteine)' below.)

●Megaloblastic versus macrocytic anemia – Both vitamin B12 and folate deficiencies cause megaloblastic anemia. Megaloblastic anemia is a term that refers to anemia in which the process of nucleic acid metabolism is impaired, resulting in nuclear-cytoplasmic dyssynchrony, reduced number of cell divisions in the bone marrow, and nuclear abnormalities in both myeloid and erythroid precursors (picture 1).

The term megaloblastic should be confined to nucleated red blood cell (RBC) precursors when used to describe red blood cell morphology. Macrocytic anemia is purely a morphologic term that describes large RBCs in the peripheral blood and includes all anemias with a high mean corpuscular volume (MCV). Megaloblastic anemia is a specific subtype of macrocytic anemia. (See "Macrocytosis/Macrocytic anemia", section on 'Megaloblastic anemia'.)

The Centers for Disease Control in the United States maintains dietary fact sheets for vitamin B12 and folate that discuss the amount of these vitamins in selected food sources as well as recommended intakes and populations at risk for deficiency. Additional information about dietary intake and absorption of these vitamins is presented separately. (See "Causes and pathophysiology of vitamin B12 and folate deficiencies", section on 'Overview of intake and metabolism'.)

EPIDEMIOLOGY — The prevalence of vitamin B12 and folate deficiencies is likely to vary among different populations and depending on the threshold used to define deficiency. Examples include the following:

●General population – In a 2016 series of 3324 patients with anemia in a general practice population in the Netherlands, 249 had macrocytosis [3]. Of these, there were 46 cases of vitamin B12 deficiency (1.4 percent of all anemic individuals; 18 percent of those with macrocytic anemia) and 16 cases of folate deficiency (0.5 percent of all anemic; 6 percent of macrocytic anemia). A 2014 examination of folate testing performed on outpatients in a single center in Boston, Massachusetts (United States) found folate deficiency in only 47 of 84,187 (0.06 percent); another 166 (0.2 percent) had low-normal values (3.0 to 3.9 ng/mL) [4].

Pernicious anemia (vitamin B12 deficiency due to autoantibodies) (see 'Terminology' above) is most common in White people from northern Europe; the incidence is lower in individuals with African or non-northern European ancestry.

●Older adults – Multiple studies of older adults have shown higher prevalences of vitamin B12 deficiency or insufficiency than in younger adults, with prevalences in older adults ranging from 5 to 14 percent [5-8].

●Hospitalized patients – In a 2015 report of hospitalized inpatients in Canada who had testing for vitamin B12 and folate levels, vitamin B12 deficiency was observed in 98 of 3154 (3.1 percent); an additional 425 individuals (13.5 percent) had vitamin B12 levels in the low normal range (138 to 221 pmol/L), the clinical manifestations of which are uncertain [9]. Folate deficiency (assessed using red blood cell folate) (see 'RBC folate' below) was observed in 4 of 2563 (0.16 percent). It should be noted that these prevalences apply to the subset of patients in whom testing was performed; the prevalences in all hospitalized patients is expected to be lower.

A report from Israel that measured serum folate in 726 patients hospitalized in the Internal Medicine Department over a one-year period found 97 (13.4 percent) to be folate deficient [10]. While the folate-deficient patients had higher in-hospital mortality than non-deficient patients (18.6 percent versus 12.1 percent), mortality did not differ between mild folate deficiency and severe folate deficiency, suggesting that deficiency occurred in the setting of underlying significant comorbidity.

●Infants and young children

•Maternal B12 deficiency – Vitamin B12 deficiency is rare in infants but may be seen in newborns and breast-fed infants of vitamin B12-deficient mothers [8]. This neonatal vitamin B12 deficiency, if not identified and treated, can result in developmental delay or permanent neurologic damage [11]. Newborn screening programs evaluate for metabolic disorders including methylmalonic acidemia and homocystinuria and can also detect vitamin B12 deficiency in newborns. In the United States, the rate of nutritional vitamin B12 deficiency from 2003 to 2007 was reported to be 0.88 in 100,000 births (1 in 113,636) [12]. However, using more sensitive and specific second-tier testing in 176,702 infants in Germany, the rate of vitamin B12 deficiency was 1 in 5355 [13]. In another study of 258,637 infants in Spain, screening identified 130 with acquired vitamin B12 deficiency (1 in 1989) [14].

•Lack of dietary folate – Folate deficiency may occur in a child unable to tolerate a normal diet or in severely malnourished infants or children. Deficiencies are also reported in infants drinking goats milk, which is deficient in folate [15,16]. (See "Micronutrient deficiencies associated with protein-energy malnutrition in children".)

The prevalence of folate deficiency in healthy people with normal dietary intake has declined progressively in countries that have implemented routine folic acid supplementation of foods (including grains, breads, cereals, pastas and rice), which began in the late 1990s and early 2000s in many parts of the world [17]. The countries that use fortification of foods with folic acid are illustrated on a website from the Food Fortification Initiative [18]. This phenomenon is illustrated by the following examples:

●In the 2015 report from Canada that evaluated hospitalized patients for folate deficiency and found a prevalence of 0.16 percent, the only causes of folate deficiency were alcohol use, a malabsorption syndrome, decreased oral intake due to schizophrenia, and a spurious value [9].

●Direct measurement of population folate levels in the NHANES study showed that after national fortification began, the mean serum folate level was 2.5 times higher than in the pre-fortification period, and red blood cell folate was 1.5 times higher [19].

In contrast, the prevalence of vitamin B12 deficiency has remained stable over time. This is likely because most cases of vitamin B12 deficiency are due to decreased absorption rather than dietary lack; therefore, routine supplementation of vitamin B12 has not been implemented, and measurement of vitamin B12 status is indicated in the evaluation of anemia.

ASSOCIATED CONDITIONS — Many individuals with vitamin B12 or folate deficiency have an associated condition that predisposes them to one or more vitamin deficiencies. In some cases, these conditions may be known, and in others they may be appreciated in retrospect after the diagnosis of vitamin B12 or folate deficiency has been made.

●Vitamin B12 deficiency – Conditions that may be associated with vitamin B12 deficiency are listed in the table (table 2). Examples include [20-23]:

•Decreased intake (eg, reduced intake of animal products, strict vegan diet, breastfeeding by a vitamin B12-deficient mother).

•Decreased absorption (eg, gastrectomy, bariatric surgery, resection of the terminal ileum, Crohn disease, celiac disease, pancreatic insufficiency, bacterial overgrowth, fish tapeworm infection, gastric atrophy associated with aging).

•Other autoimmune conditions, such as thyroid disease or vitiligo, in individuals with pernicious anemia.

•Medications and drugs that interfere with absorption or stability (eg, metformin, histamine receptor antagonists, proton pump inhibitors, nitrous oxide). (See "Causes and pathophysiology of vitamin B12 and folate deficiencies", section on 'Metformin'.)

•Rare genetic disorders. (See "Causes and pathophysiology of vitamin B12 and folate deficiencies".)

Annual monitoring for vitamin B12 deficiency is recommended for patients receiving metformin; this is discussed in more detail separately. (See "Metformin in the treatment of adults with type 2 diabetes mellitus", section on 'Monitoring'.)

●Folate deficiency – Conditions that may be associated with folate deficiency are listed in the table (table 3). Examples include [2]:

•Increased requirements due to pregnancy, hemolytic anemia.

•Decreased intake, especially in individuals with excessive alcohol use and corresponding reductions in dietary intake of folate-rich foods such as fresh vegetables and fortified grains.

•Use of goat's milk (which has low folate concentrations) as the main source of food in infants and toddlers.

•Residence in a place where routine folate supplementation of foods does not occur.

•Decreased absorption in the setting of gastric bypass surgery.

•Loss during hemodialysis (along with other water-soluble vitamins), although patients are routinely given a multivitamin containing folic acid. (See "Pathogenesis and treatment of malnutrition in patients on maintenance hemodialysis".)

•Increased requirements, such as occur with severe chronic hemolytic anemia or exfoliative dermatitis.

•Medications and drugs that interfere with metabolism (eg, methotrexate, sulfasalazine). (See "Major side effects of low-dose methotrexate", section on 'Myelosuppression'.)

•Rare genetic disorders. (See "Causes and pathophysiology of vitamin B12 and folate deficiencies".)

Bariatric surgery places patients at risk of developing both vitamin B12 and folate deficiencies (along with other deficiencies), requiring life-long dietary supplementation. Bariatric surgery can also lead to iron deficiency, which causes microcytic anemia that may obscure the finding of macrocytosis on the CBC (see 'CBC and blood smear' below). Specific bariatric procedures have different risks depending on whether the absorptive surfaces were removed. As an example, Roux-en-Y bypass is more likely to cause vitamin B12 deficiency than other procedures. Routine vitamin supplementation is recommended, as shown in the table and discussed separately (table 4). (See "Bariatric surgery: Postoperative nutritional management".)

The mechanisms by which these conditions cause deficiencies are discussed in more detail separately. (See "Causes and pathophysiology of vitamin B12 and folate deficiencies".)

CLINICAL PRESENTATION

Typical presentation — Patients with vitamin B12 and/or folate deficiency most commonly present with anemia, which may be associated with nonspecific symptoms (often fatigue, which can occur in anemia of any etiology). Another common presentation is with an incidental finding of mild anemia. In settings with frequent complete blood count (CBC) testing, most patients will not have the classic findings previously associated with these deficiencies, such as major neurologic changes with vitamin B12 deficiency.

The classic findings associated with vitamin B12 and folate deficiency include worsening macrocytic anemia, yellowed skin (caused by combined anemia and jaundice), and variable neurologic abnormalities more prominent in vitamin B12 deficiency (cognitive slowing and neuropathy).

These were seen more commonly in the early 20^th century when deficiencies were often quite advanced upon presentation. These findings may be present, but often more subtle presentations are seen in healthy individuals living in resource-rich countries. Routine supplementation of grains with folic acid and routine CBC testing have made dietary deficiencies less common and have led to earlier diagnosis in many individuals.

Macrocytic anemia — The presence of symptoms attributable to anemia depends on the rate that deficiency has developed, the severity of the deficiency, the hemoglobin level, and the person's overall health [23]. Many individuals with vitamin B12 or folate deficiency have vague or nonspecific symptoms (fatigue, irritability, cognitive decline), which are likely to be due at least in part to anemia. This is because red blood cell (RBC) precursors in the bone marrow divide rapidly and thus are sensitive to the lack of nucleic acids that occurs when these vitamins are deficient. (See "Causes and pathophysiology of vitamin B12 and folate deficiencies".)

In the era of frequent CBC testing, many deficiencies come to medical attention as an incidental finding of anemia and/or macrocytosis. Individuals with vitamin B12 deficiency can also develop pancytopenia. (See 'CBC and blood smear' below.)

Symptoms attributable to tissue hypoxia and organ ischemia (eg, chest pain, shortness of breath) may occur if the anemia is severe and/or the individual has underlying ischemic heart disease. However, the anemia typically develops gradually in vitamin B12 deficiency, and compensatory mechanisms may mitigate symptoms related to tissue hypoxia, such as palpitations, light-headedness, and shortness of breath [23]. Individuals with more severe anemia may have skin pallor. When combined with mild jaundice due to hemolysis, this may cause the skin to be a "peculiar lemon-yellow color" [23].

The laboratory characteristics of the anemia are discussed below. (See 'CBC and blood smear' below.)

Gastrointestinal symptoms — Vitamin B12 deficiency can cause glossitis (including pain, swelling, tenderness, and loss of papillae and/or hyperpigmentation of the tongue), and folate deficiency can cause oral ulcers [24]. This occurs because cells in the gastrointestinal tract divide rapidly and are thus sensitive to the lack of nucleic acids that occurs when these vitamins are deficient.

Other individuals may have gastrointestinal symptoms related to the underlying condition that caused the deficiency, such as pain or diarrhea related to inflammatory bowel disease, celiac disease, or other malabsorptive states. The mechanisms by which the deficiencies cause gastrointestinal symptoms and the underlying conditions cause the deficiencies are presented separately. (See "Causes and pathophysiology of vitamin B12 and folate deficiencies".)

Neuropsychiatric changes — Neuropsychiatric manifestations may be present in both vitamin B12 and folate deficiencies. Although these findings are most commonly ascribed to vitamin B12 deficiency, neurocognitive and other changes have been reported with folate deficiency as well [2,25-29]. The mechanism is discussed separately. (See "Causes and pathophysiology of vitamin B12 and folate deficiencies" and "Evaluation of cognitive impairment and dementia".)

The most common neurologic findings in vitamin B12 deficiency are symmetric paresthesias or numbness and gait problems [20,30]. The neuropathy is typically symmetric and affects the legs more than arms. The classic neurologic finding in vitamin B12 deficiency is subacute combined degeneration of the dorsal (posterior) and lateral columns (white matter) of the spinal cord due to demyelination. It is associated with progressive weakness, ataxia, and paresthesias that may progress to spasticity and paraplegia. These findings are helpful diagnostically if present but may not occur in all cases, especially if diagnosed earlier in the course of the deficiency. The neurologic features of spinal cord disorders are discussed in more detail separately. (See "Disorders affecting the spinal cord".)

Other findings may include one or more of the following [2,20,31-34]:

●Depression or mood impairment

●Irritability

●Insomnia

●Cognitive slowing

●Forgetfulness

●Dementia

●Psychosis

●Visual disturbances, which may be associated with optic atrophy

●Peripheral sensory deficits

●Weakness, which may progress to paraplegia and incontinence if severe

●Impaired position sense

●Impaired vibration sense

●Lhermitte sign, a shock-like sensation that radiates to the feet during neck flexion

●Ataxia or positive Romberg test

●Abnormal deep tendon reflexes

●Extrapyramidal signs (eg, dystonia, dysarthria, rigidity)

●Restless legs syndrome

Nonspecific fatigue may be a presenting symptom even in the absence of anemia, but fatigue is usually associated with some of the findings above, particularly depression/mood impairment or cognitive slowing. Although evaluation for fatigue might include evaluation of vitamin B12 level, we do not recommend supplementation unless vitamin B12 levels are low. (See "Treatment of vitamin B12 and folate deficiencies", section on 'Vitamin B12'.)

In individuals with vitamin B12 deficiency, neuropsychiatric symptoms can be present even in the absence of anemia or macrocytosis, and the lack of these hematologic changes cannot be used to exclude vitamin B12 deficiency as a cause of neuropsychiatric symptoms. This was illustrated in a study that assessed hematologic findings in 141 consecutive patients with neuropsychiatric symptoms caused by vitamin B12 deficiency [26]. Of these, 40 (28 percent) had a normal hematocrit, MCV, or both. Vitamin B12 replacement led to a clinical response in all evaluable patients.

Retrospective studies have suggested a correlation between vitamin B12 levels and declining cognitive function over time, but a causal relationship has not been demonstrated [35]. (See "Evaluation of cognitive impairment and dementia", section on 'Laboratory testing' and "Prevention of dementia", section on 'Vitamins B6, B12, and folate'.)

Neuroimaging is generally not performed, but if done as part of the evaluation for other conditions, magnetic resonance imaging (MRI) of the spinal cord in an individual with vitamin B12 deficiency may show classic changes described as "an inverted V-shaped pattern" in the cervical and thoracic spinal cord [20,33,36]. The localization of some of the specific neurologic manifestations (eg, Lhermitte sign, related to changes in the cervical spinal cord) is discussed in more detail separately. (See "Anatomy and localization of spinal cord disorders".)

Subtle neurologic, cognitive, or psychiatric changes are among of the most commonly encountered symptoms in primary care practice, particularly in older individuals. A full discussion of the complex issues related to these findings and their possible causes is presented in detail in separate topic reviews. (See "Evaluation of cognitive impairment and dementia" and "Unipolar depression in adults: Assessment and diagnosis" and "Approach to the patient with sensory loss" and "Overview of cerebellar ataxia in adults".)

Infants and maternal vitamin B12 deficiency — Vitamin B12 is transferred to the fetus during gestation and via breast milk after birth. Thus, an infant born to a mother with vitamin B12 deficiency can be deficient at birth and at risk for further deficiency if exclusively breast-fed.

Vitamin B12 deficiency in an infant may present with pancytopenia and/or macrocytosis; there may be associated developmental delay or regression, feeding difficulties, hypotonia, irritability, tremors, or convulsions [20,37].

Newborn screening may be able to identify infants with vitamin B12 deficiency at birth. (See 'Epidemiology' above.)

Other rare features — Although rarely the presenting complaint, the following have also been associated with vitamin B12 and/or folate deficiency:

●Vitamin B12 – Other findings in vitamin B12 deficiency:

•Skin – Skin hyperpigmentation and hypopigmentation can occur [20,31,38]. Case reports have described hyperpigmentation on the hands (picture 2) and feet (picture 3) that resolved after vitamin B12 repletion [38]. Reports have also described maculopapular skin lesions that respond to vitamin B12 supplementation [39].

•Bones – Increased risk of osteoporosis and fractures of the hip or spine (mechanism unknown, possibly due to suppression of osteoclast activity) [40-46]. These data are observational, and some studies have not observed the association [47].

•Cancer – Increased risk of gastric cancer in individuals with pernicious anemia. (See 'Determining the underlying cause of vitamin B12 deficiency' below.)

●Folate – Other findings in folate deficiency:

•Cancer – Possible increased risk of certain malignancies (eg, colon cancer), but the evidence is not strong, and there is possible increased risk with excess folic acid supplementation. (See "Epidemiology and risk factors for colorectal cancer", section on 'Folic acid and folate'.)

•Neural tube defects – Folate deficiency during embryogenesis is known to increase the risk of neural tube defects, as discussed in detail separately. (See "Neural tube defects: Overview of prenatal screening, evaluation, and pregnancy management", section on 'Folate deficiency' and "Preconception and prenatal folic acid supplementation".)

Distinguishing vitamin B12 from folate deficiency — There are no presenting features that can definitively distinguish between vitamin B12 and folate deficiencies. However, the typical clinical presentations differ as follows:

●Diet – Vitamin B12 deficiency may be seen in strict vegans or vegetarians who do not take supplemental vitamin B12. Individuals living in resource-rich settings who are able to take in a normal diet are extremely unlikely to become folate deficient. Folate deficiency may be seen in individuals with a history of markedly decreased dietary intake or excessive alcohol use, even without other signs of malnutrition [48].

●Associated clinical findings – The classical clinical picture of subacute combined degeneration of the cord (eg, progressive weakness, ataxia, and paresthesias that can advance to spastic paraplegia) is confined to patients with vitamin B12 deficiency. A variety of neuropsychiatric changes have been ascribed to both vitamin B12 and folate deficiency as discussed above; however, because of the widespread prevalence of neuropsychiatric disorders in the general population and the lower frequency of symptomatic vitamin B12 and folate deficiencies, an informed approach to evaluation of neuropsychiatric changes is warranted. (See 'Neuropsychiatric changes' above and 'Other rare features' above.)

●Time course – The time course of developing the deficiency may be helpful, if known.

•Vitamin B12 deficiency typically develops over the course of years, as total body stores are large (approximately 3 to 5 mg, which in many cases is sufficient to provide adequate levels of vitamin B12 for 5 to 10 years) [49]. However, in infants born to vitamin B12-deficient mothers, there may not be any stores established, and the infant may be born deficient. Another exception is exposure to nitrous oxide, which causes rapid depletion of vitamin B12 [50,51].

•Folate deficiency can develop rapidly (weeks to months, depending on baseline stores) as body stores are limited (approximately 5 to 10 mg) and become rapidly depleted during normal cell division.

DIAGNOSTIC EVALUATION

Overview of evaluation — In countries in which dietary deficiency is less of a concern, vitamin B12 and/or folate deficiency may be suspected in a patient with one or more of the following (algorithm 1):

●Unexplained anemia, macrocytosis (mean corpuscular volume [MCV] >100 fL) (picture 4), pancytopenia, or hypersegmented neutrophils (picture 5).

●Unexplained neurologic or psychiatric symptoms.

●Strict vegan diet or conditions that may interfere with absorption. (See 'Associated conditions' above.)

●Certain autoimmune disorders such as thyroiditis or vitiligo, or those taking chronic metformin therapy.

●Gastrointestinal symptoms such as sore tongue, anorexia, or diarrhea [22].

Periodic monitoring of vitamin B12 levels may be appropriate in individuals with intestinal disorders that might affect absorption of vitamin B12, including celiac disease, inflammatory bowel disease, small intestinal bacterial overgrowth, ileal resection, radiation enteritis, and chronic metformin therapy. Information on which tests to order and the frequency of monitoring is discussed in topic reviews on these disorders.

Medications associated with lower vitamin B12 levels including metformin and proton pump inhibitors should be assessed. (See 'Associated conditions' above.)

For an individual with suspected vitamin B12 or folate deficiency, the history should include questions about previously diagnosed associated conditions, particularly celiac disease or inflammatory bowel disease; bariatric, gastric, or intestinal surgery; reduced dietary intake (eg, vegan or vegetarian diet, lack of fresh vegetables); alcohol use (as an independent cause of macrocytic anemia and as a possible predictor of reduced dietary intake); and any symptoms, including subtle neurologic or psychiatric symptoms, such as those described above [20,34]. (See 'Clinical presentation' above and "Causes and pathophysiology of vitamin B12 and folate deficiencies".)

The physical examination should focus on gastrointestinal and dermatologic findings, as well as hepatosplenomegaly and lymphadenopathy, which may suggest other causes of macrocytic anemia (table 5). A thorough neurologic examination should be conducted for signs of altered affect or mentation and/or findings associated with central or peripheral neuropathy (eg, impaired sense of vibration, proprioception, or light touch, ataxia, weakness) [20]. These other causes are discussed below. (See 'Differential diagnosis' below.)

We test for both deficiencies when evaluating individuals with gastrointestinal conditions known to affect absorption of both vitamins (algorithm 1). In the absence of these gastrointestinal abnormalities in individuals who are eating a healthy diet that includes food fortified with folic acid, and in the absence of other reasons to suspect folate deficiency, we only test for vitamin B12 deficiency. The specific tests required (and their sequence) depend on the clinical picture, findings on the complete blood count (CBC), and the results of initial testing for the vitamin B12 (and folate level if appropriate), as described in the following sections. (See 'Laboratory testing' below.)

Our approach is consistent with a 2014 guideline from the British Committee for Standards in Haematology [24]. Links to this and other guidelines are presented separately. (See 'Society guideline links' below.)

Routine population screening for vitamin B12 deficiency has been debated, but there is no evidence that this improves outcomes or is cost effective, and we do not screen otherwise healthy individuals without clinical findings or risk factors for deficiency. However, we suggest a lower threshold for testing populations at increased risk of deficiency due to the clinical factors discussed above, as well as older adults in general if they develop signs or symptoms indicative of deficiency or as part of the evaluation of cognitive decline. (See 'Associated conditions' above and 'Epidemiology' above.)

Laboratory testing

CBC and blood smear — Deficiencies of vitamin B12 and folate can both cause megaloblastic anemia. The findings on the CBC and the blood cell morphology on the peripheral blood smear are identical with both deficiencies and may include one or more of the following:

●Anemia

●Macrocytic red blood cells (RBCs; eg, mean corpuscular volume [MCV] >100 fL) or macro-ovalocytosis (picture 4)

●Mild leukopenia and/or thrombocytopenia

●Low reticulocyte count

●Hypersegmented neutrophils (picture 5) on the peripheral blood smear (ie, >5 percent of neutrophils with ≥5 lobes or ≥1 percent of neutrophils with ≥6 lobes)

Macrocytosis and hypersegmentation of neutrophils precede the development of anemia and may be present as isolated findings (without anemia). The mechanisms by which RBCs become macrocytic and neutrophils become hypersegmented (both features of megaloblastic anemia) are discussed separately. (See "Macrocytosis/Macrocytic anemia", section on 'Megaloblastic anemia' and "Causes and pathophysiology of vitamin B12 and folate deficiencies", section on 'Hematopoiesis'.)

On rare occasions, severe acquired vitamin B12 deficiency or heritable gene variants affecting cobalamin metabolism may be associated with schistocytes and may resemble a thrombotic microangiopathy [52]. (See "Diagnostic approach to suspected TTP, HUS, or other thrombotic microangiopathy (TMA)", section on 'Exclude systemic disorders associated with MAHA and thrombocytopenia' and "Diagnostic approach to suspected TTP, HUS, or other thrombotic microangiopathy (TMA)", section on 'Overview of primary TMA syndromes'.)

An MCV value >115 fL is more specific to vitamin B12 or folate deficiency than other conditions in the differential diagnosis such as hypothyroidism or myelodysplastic syndrome. However, a normal MCV does not exclude vitamin B12 or folate deficiency. In a 1994 series of 100 consecutive patients with a confirmed diagnosis of vitamin B12 deficiency based on the presence of autoantibodies to intrinsic factor (IF), a positive Schilling test, or both, only 29 percent had anemia and only 36 percent had an MCV >100 fL [53]. In a cohort of healthy older adults (age 67 or older) who participated in the Framingham heart study, most of the individuals who were found to have laboratory values of low vitamin B12 or folate on population screening had a normal MCV [5].

The MCV does not help distinguish vitamin B12 deficiency from folate deficiency. Further, if a patient has concomitant microcytosis due to iron deficiency or thalassemia, the macrocytosis may be masked on a CBC because the MCV reflects the average volume of all RBCs. However, in this instance (combined macrocytosis and microcytosis), the red cell distribution width (RDW) may be increased, and a blood smear may reveal both small and large red blood cells [54]. Other forms of anemia may also blunt the macrocytosis caused by vitamin B12 or folate deficiency.

In addition to affecting the morphology of mature RBCs and neutrophils, megaloblastic anemia can cause premature destruction of developing RBCs in the bone marrow (ineffective erythropoiesis leading to intramedullary hemolysis) and the peripheral circulation (hemolysis). (See "Causes and pathophysiology of vitamin B12 and folate deficiencies".)

Laboratory findings associated with hemolysis may be present, including increased lactate dehydrogenase (LDH; increases may be dramatic with extremely high LDH levels), increased indirect bilirubin, and low haptoglobin. Unlike other forms of hemolytic anemia, the reticulocyte count in vitamin B12 and folate deficiency is also low (or normal), reflecting reduced blood cell production.

Serum vitamin B12 and folate levels — For individuals with typical findings on the CBC and a low reticulocyte count, the only initial testing needed is a serum vitamin B12 and, in some cases, a folate level [20,24]. We often omit the folate level if the individual is consuming a varied diet containing folate-supplemented grains and has normal gastrointestinal anatomy and function, as folate deficiency in these individuals is rare (algorithm 1). We test both vitamin B12 and folate levels in individuals with gastrointestinal conditions, excess alcohol use, or dietary patterns known to cause both deficiencies. (See 'Epidemiology' above.)

Most individuals can be determined to have normal or deficient levels using laboratory-specific cutoff values provided by the testing laboratory with the patient's results. For those with a clearly normal or deficient level, additional testing to diagnose deficiency is unlikely to be required [20,24,55]. For those with borderline levels, additional testing can be used to determine the clinical significance. (See 'Metabolite testing (MMA and homocysteine)' below.)

Additional postdiagnostic testing to determine the underlying cause of the deficiency and/or other associated medical conditions is discussed below. (See 'Post-diagnostic testing' below.)

Vitamin B12 normal ranges — The following reflects typical serum vitamin B12 levels and their interpretation [55,56]:

●Above 300 pg/mL (above 221 pmol/L) – Normal; deficiency unlikely (sensitivity of approximately 90 percent; however, the assay may not be as sensitive in individuals with anti-intrinsic factor [IF] antibodies) [20].

●200 to 300 pg/mL (148 to 221 pmol/L) – Borderline; deficiency is possible and additional testing is useful. (See 'Additional testing for selected individuals' below.)

●Below 200 pg/mL (below 148 pmol/L) – Low; consistent with deficiency. Additional testing may be appropriate to determine the accuracy of the diagnosis and possibly its cause (see 'Determining the underlying cause of vitamin B12 deficiency' below). Treatment is discussed separately. (See "Treatment of vitamin B12 and folate deficiencies", section on 'Vitamin B12'.)

If the level is clearly normal and another cause of macrocytosis, anemia, and/or other clinical features has been identified (see 'Differential diagnosis' below), we do not perform further testing. For patients with fatigue or other neuropsychiatric symptoms and either normal vitamin B12 levels or low/borderline B12 levels with normal results on metabolite testing, there is no evidence that vitamin B12 deficiency is responsible or that treatment corrects fatigue. Patients may request injections based on material they access in the lay press; if this happens, we counsel patients that there is no medical rationale for that approach [57,58]. (See "Treatment of vitamin B12 and folate deficiencies", section on 'Individuals without documented vitamin B12 deficiency'.)

If the level is clearly deficient, we confirm and treat the deficiency and determine the underlying cause, which has implications for the route of replacement and the duration of therapy, as discussed below. (See 'Basis for confirmed diagnosis' below and 'Determining the underlying cause of vitamin B12 deficiency' below.)

In most laboratories, serum vitamin B12 levels are measured using a competitive chemiluminescence assay that has an estimated sensitivity of approximately 95 percent in symptomatic patients (a specificity of approximately 80 percent or less) [59]. The assay uses binding to intrinsic factor (IF) following dissociation from binding proteins, with a readout based on the amount of unbound IF remaining.

There are several important caveats regarding the performance of automated tests, and assays by different laboratories using different methods often show poor agreement. Additional testing is warranted if levels are in the borderline range or if the results of laboratory testing are discordant with the clinical picture (eg, patient with chronic or worsening macrocytic anemia for which all other testing is unrevealing). This testing is discussed below. (See 'Additional testing for selected individuals' below.)

Examples of the caveats related to vitamin B12 testing include [20,34,56,59-61]:

●Normal ranges can vary based on the characteristics of the assay used for testing. In a retrospective record review of 456 ambulatory individuals, vitamin B12 levels fluctuated by a median of 23 percent over a period of weeks [62]. One-fifth of the individuals had variations greater than 100 pg/mL.

●Tissue stores of vitamin B12 are apparently only moderately correlated with serum vitamin B12 levels, possibly due to variations in serum vitamin B12 binding proteins.

Causes of spuriously low vitamin B12 — Vitamin B12 circulates in plasma bound to haptocorrin (TCI; also called R-factor) and transcobalamin II. Approximately 80 percent of vitamin B12 is bound to haptocorrin and is functionally inactive. Vitamin B12 bound to transcobalamin II (holotranscobalamin) is the active form absorbed by cells.

Decreased production of vitamin B12 binding proteins has been proposed to be responsible for the spuriously low values, as levels of the active holotranscobalamin were unchanged in a series of individuals with decreased vitamin B12 levels [63]. The effect of vitamin B12 binding proteins on levels of functional vitamin B12 is illustrated in the figure (figure 4).

Certain conditions or medications may be associated with spuriously low serum vitamin B12 levels and thus might cause the appearance of vitamin B12 deficiency when the patient is not deficient. Examples include:

●Multiple myeloma

●HIV infection

●Pregnancy

●Oral contraceptives

●Diphenylhydantoin

An example of spuriously low vitamin B12 level in pregnancy was demonstrated in a series of 50 pregnant individuals with low vitamin B12 levels (45 to 199 pg/mL), in whom metabolite testing for methylmalonic acid (MMA) and homocysteine showed no correlation with vitamin B12 level. (See 'Metabolite testing (MMA and homocysteine)' below.)

Causes of spuriously high vitamin B12 — Certain conditions may be associated with spuriously increased vitamin B12 levels and thus might cause the appearance of normal vitamin B12 levels when the patient is deficient. Examples include [23]:

•Occult malignancy

•Myeloproliferative neoplasm

•Alcoholic liver disease

•Kidney disease

•Certain inborn errors of metabolism

•Nitrous oxide exposure (which can also cause vitamin B12 deficiency, as evidenced by clinical symptoms and high methylmalonic acid [MMA] levels) [64]

In addition, autoantibodies to IF in individuals with pernicious anemia may compete with IF in the chemiluminescence assay and result in spuriously normal vitamin B12 levels [65-67]. If the vitamin B12 level is very high (eg, 800 pg/mL), we do not worry about this effect in the absence of clinical features suggesting vitamin B12 deficiency; however, if the vitamin B12 level is borderline or low normal and/or other clinical features suggest vitamin B12 deficiency (see 'Clinical presentation' above), it is prudent to obtain other testing such as MMA and homocysteine levels. (See 'Additional testing for selected individuals' below.)

Folate normal ranges — When indicated, routine testing for folate deficiency is usually done using the serum folate level.

The following reflects typical serum folate levels and their interpretation [55,56]:

●Above 4 ng/mL (above 9.1 nmol/L) – Normal. Suggests folate is not deficient, unless the individual has recently consumed a folate-containing meal or supplement. In such cases, RBC folate can be obtained (see 'RBC folate' below), although we generally prefer metabolite testing (see 'Metabolite testing (MMA and homocysteine)' below). The RBC folate level is not needed in routine testing because it does not provide additional information and is more costly to obtain.

●From 2 to 4 ng/mL (from 4.5 to 9.1 nmol/L) – Borderline. Additional testing may be indicated depending on the clinical circumstances and the degree of suspicion for folate deficiency. (See 'Additional testing for selected individuals' below.)

●Below 2 ng/mL (below 4.5 nmol/L) – Low. Consistent with folate deficiency. (See 'Basis for confirmed diagnosis' below.)

Values may be slightly higher in the first six months of life. For hospitalized patients, blood samples should be obtained immediately on admission, before any meals have been consumed and before any blood transfusions have been given, as even a single meal or transfusion may normalize serum folate levels for several weeks even if deficiency is present.

Additional testing for selected individuals

Metabolite testing (MMA and homocysteine) — Additional testing for intermediates in vitamin B12 and folate metabolism (methylmalonic acid [MMA] and homocysteine) can be reserved for cases in which initial test results for vitamin B12 and/or folate levels are borderline (near the lower limit of normal) or inconclusive, or if clinical findings are discordant with initial testing values (eg, low-normal vitamin B12 level in an individual with unexplained macrocytic anemia or unexplained neurologic findings) (algorithm 1).

Because of concerns regarding the accuracy of vitamin B12 testing, in most cases of suspected deficiency, we often confirm the diagnosis by testing for the intermediates methylmalonic acid [MMA] and homocysteine [68,69]. Ideally, at least two biochemical abnormalities should be sought to make the diagnosis in individuals who are asymptomatic since an isolated abnormality, such as elevated MMA, may be spurious [49].

The normal ranges for MMA and homocysteine are laboratory-dependent; laboratory-specific and assay-specific cutoffs should be used. Examples of typical normal ranges are: MMA 70 to 270 nmol/L; homocysteine 5 to 15 micromol/L. For MMA, the American Board of Internal Medicine uses 0 to 400 nmol/L (0 to 0.40 micromol/L). (See "Laboratory test reference ranges in adults", section on 'Methylmalonic acid (MMA), serum'.)

Interpretation is as follows:

●MMA and homocysteine normal – No deficiency of vitamin B12 or folate.

●MMA and homocysteine elevated – Deficiency of vitamin B12 (does not eliminate the possibility of folate deficiency).

●MMA normal, homocysteine elevated – No deficiency of vitamin B12. Consistent with deficiency of folate.

MMA is elevated in vitamin B12 deficiency but not in folate deficiency. This is because vitamin B12 is a cofactor in conversion of methylmalonyl-CoA to succinyl-CoA, a reaction that occurs in mitochondria and is catalyzed by methylmalonyl-CoA mutase [59]. In the absence of vitamin B12, this reaction cannot proceed normally, and MMA accumulates (figure 3).

Urinary MMA can also be measured, although this is not done in routine practice [7].

MMA can be elevated in the absence of vitamin B12 deficiency in individuals with chronic kidney disease. Other alternatives for testing in this setting include measuring autoantibodies to intrinsic factor and examining the clinical response to administration of parenteral vitamin B12. For individuals with a low serum vitamin B12 level and a high MMA, we test for autoantibodies to intrinsic factor to establish whether pernicious anemia (PA) is the underlying cause. (See 'Autoantibodies to intrinsic factor' below and 'Response to vitamin replacement' below.)

The sensitivity of MMA and homocysteine for vitamin B12 deficiency was addressed in a 1994 study that measured these metabolites in a series of 406 individuals diagnosed with vitamin B12 deficiency based on a low vitamin B12 level (<200 pg/mL) plus a clinical finding (diagnostic bone marrow, blood smear, and response to vitamin B12 administration) [70]. Most of the patients had PA. Of these 406 individuals, 94.5 percent had elevations of both metabolites, and all but one had an increase in at least one metabolite, with a sensitivity of 99.8 percent for the diagnosis of vitamin B12 deficiency.

These findings were very useful for characterizing the metabolite patterns in individuals with obvious clinical deficiency, and they suggest that metabolite testing has a high sensitivity in people with PA who have overtly low serum vitamin B12 levels and associated clinical features. The sensitivity and specificity of this testing in individuals with borderline vitamin B12 levels and/or the absence of clinical features, where metabolite testing would be most useful, is not as clear.

Homocysteine is elevated in both vitamin B12 and folate deficiencies. This is because both vitamins are required for the metabolism of homocysteine to methionine (figure 3). In the absence of either vitamin, this process cannot occur normally, and homocysteine accumulates. Thus, a normal MMA and an increased homocysteine level is consistent with folate deficiency. In the 1994 study that measured metabolites in individuals with clinically obvious deficiencies, homocysteine was increased in 91 percent of those with folate deficiency (based on a low folate level [<4 ng/mL], normal vitamin B12 level [>300 pg/mL], and presence of an underlying disorder associated with folate deficiency) [70].

There may be substantial fluctuations in the measured MMA and homocysteine levels, and unexpectedly normal or abnormal levels should be repeated [62,71]. Conditions other than vitamin B12 and folate deficiency that have the potential to alter the MMA include the following:

●Chronic kidney disease (increased MMA levels).

●Methylmalonic acidemia/aciduria (increased MMA levels). (See "Methylmalonic acidemia".)

●Hereditary homocysteinemia (increased homocysteine levels). (See "Overview of homocysteine".)

●Antibiotic treatment (lower MMA levels in two case reports, possibly due to alterations in intestinal flora that led to a lack of the MMA precursor propionic acid) [69].

In such cases, it may be necessary to test for autoantibodies associated with PA or to determine the hematologic response to vitamin administration. (See 'Autoantibodies to intrinsic factor' below and 'Response to vitamin replacement' below.)

Autoantibodies to intrinsic factor — Testing for autoantibodies to intrinsic factor (IF) is used to identify pernicious anemia (PA), an autoimmune condition that leads to severely impaired vitamin B12 absorption. We test for anti-IF antibodies in individuals who have biochemical evidence of vitamin B12 deficiency (low serum vitamin B12 level and/or high MMA level) without another obvious cause. The testing is typically done with an immunoassay. The sensitivity of IF autoantibodies is relatively low. As an example, in a 1992 study of 324 patients with documented PA, autoantibodies to IF were present in 228 (70 percent) [72]. The specificity of autoantibodies to IF is high; if present, anti-IF autoantibodies are considered confirmatory for a diagnosis of PA [23].

Many patients with PA have autoantibodies to parietal cells; however, antiparietal cell antibodies are also seen in some patients with gastritis and are not diagnostic of PA [23].

If PA is strongly suspected and testing for autoantibodies to IF is negative, we may make the diagnosis based on other findings such as clinical manifestations and laboratory findings consistent with vitamin B12 deficiency (high MCV, hypersegmented neutrophils), especially if these findings resolve with vitamin B12 administration. Such findings would be sufficient to continue lifelong treatment. (See "Treatment of vitamin B12 and folate deficiencies", section on 'Treatment of vitamin B12 deficiency'.)

In selected cases, it may be possible to measure serum gastrin levels or pepsinogen I and II levels, although elevated gastrin and low ratio of pepsinogen I to II are also not highly specific for PA [73,74].

RBC folate — RBC folate is a surrogate for tissue folate levels. RBC folate provides information about folate status over the lifetime of RBCs, similar to hemoglobin A1C for blood glucose levels.

We no longer use this test; rather, if the serum folate is borderline and there is clinical suspicion, we use metabolite testing and/or response to folate repletion (See 'Metabolite testing (MMA and homocysteine)' above and 'Response to vitamin replacement' below.)

Some clinicians consider it to be helpful to test for RBC folate if results of serum folate testing are normal or borderline low in an individual with a strong clinical suspicion for folate deficiency (see 'Folate normal ranges' above). This especially applies if they have just eaten a folate-containing food, such as a hospital snack or meal. However, studies comparing serum folate with RBC folate have found that addition of RBC folate to serum folate testing does not provide substantially more clinical information [75,76].

Variation in the use of the RBC folate assay was illustrated in a 2003 benchmarking survey of hematology laboratories in the United Kingdom, which found that 42 percent of laboratories used serum folate, 45 percent used RBC folate, and 13 percent used both [75]. This review suggested that approximately 5 percent of people with normal serum folate levels may have evidence of folate deficiency using RBC folate testing.

An RBC folate level below 150 ng/mL (<150 mcg/L; <340 nmol/L) is consistent with folate deficiency as long as there is not concomitant vitamin B12 deficiency (RBC folate is lower in individuals with vitamin B12 deficiency) [24].

Response to vitamin replacement — Another option for individuals who have borderline laboratory values, conditions that interfere with measurement (eg, kidney disease), or discordance between laboratory testing and clinical picture is to provide replacement therapy for the vitamin suspected to be deficient (eg, parenteral vitamin B12 1000 mcg weekly for three weeks, or oral or parenteral folic acid 1 mg daily for one to two weeks).

The hematologic response can be assessed by measuring the reticulocyte count and CBC before and approximately two weeks after supplementation. Both the reticulocyte count and hemoglobin level are expected to increase as hematopoiesis improves. There will no longer be hypersegmented neutrophils on the blood smear, and the MCV will eventually return to normal, although it may remain high initially (during reticulocytosis) since reticulocytes are larger than mature RBCs.

Measuring hematologic parameters is likely to be more accurate than general well-being or neurologic symptoms since many individuals report the phenomena of more energy following a vitamin B12 shot even if they were not deficient.

Vitamin B12 administration is extremely safe and inexpensive. However, there are potential problems with using the response to vitamin B12 administration as a diagnostic test. (See 'Basis for confirmed diagnosis' below.)

Alternatively, there may be comorbidities such as anemia of chronic disease/anemia of inflammation (ACD/AI), that prevent a full reticulocyte response. However, the resolution of hypersegmented neutrophils on the peripheral blood smear is correlated to correction of the deficiency.

Folic acid administration is also nontoxic and inexpensive, but administration of folic acid alone in an individual with concurrent vitamin B12 deficiency may be associated with progression or worsening of neurologic abnormalities. Thus, it is prudent to check the vitamin B12 level, as discussed below. (See 'Other contributing factors to anemia' below.)

Schilling test (historical interest) — The Schilling test is primarily of historical interest; this test is no longer routinely available.

Prior to the availability of other testing for pernicious anemia, the Schilling test was used to determine if an individual with vitamin B12 deficiency was able to absorb vitamin B12 via the oral route. The test involved an oral dose of radiolabeled vitamin B12, with or without another substance to promote absorption, such as intrinsic factor, and a large parenteral dose of unlabeled vitamin B12 to saturate the vitamin B12 binding proteins and lead to massive excretion of the excess vitamin B12. If radiolabeled vitamin B12 was absorbed, it would be excreted in urine.

The Schilling test may be falsely normal in patients with achlorhydria who are unable to extract vitamin B12 from food but may be able to absorb it from a pill [77].

Lack of access to the radiolabeled vitamin B12 used in the test, declining expertise, and increased availability of other testing with equal or better accuracy has made the Schilling test obsolete. Alternative tests for vitamin B12 absorption are under investigation [23].

Bone marrow (generally not indicated) — A bone marrow aspirate and biopsy is not used to evaluate for vitamin B12 or folate deficiencies, and the findings on bone marrow morphology typically do not help to distinguish these deficiencies from other hematologic disorders or from each other. However, if a bone marrow evaluation is performed in an individual with vitamin B12 or folate deficiency (eg, for another suspected condition, such as myelodysplastic syndrome [MDS]), it is likely to reveal a markedly hypercellular marrow with megaloblastic erythroid hyperplasia, giant metamyelocytes, and frequent mitoses (picture 6 and picture 1). (See 'Differential diagnosis' below.)

Bone marrow abnormalities due to vitamin B12 or folate deficiency resolve rapidly after treatment is initiated.

Basis for confirmed diagnosis — We consider the diagnosis of vitamin B12 or folate deficiency to be confirmed in the following scenarios:

●Serum vitamin level(s) in the "low" range, especially if there are clinical findings consistent with deficiency.

●Elevated serum MMA and/or homocysteine levels consistent with deficiency and serum vitamin levels in the intermediate range.

●Hematologic response to administration of the deficient vitamin, as long as the response cannot be explained by another intervention. Based on concerns outlined above, this is generally used as supportive evidence and typically is not the sole basis for confirming the diagnosis. (See 'Response to vitamin replacement' above.)

We do not interpret lack of a response to administration of vitamin B12 or folate to indicate lack of deficiency, because there may be other causes of the clinical findings and/or reasons for lack of hematologic response. In such cases, we provide parenteral replacement if the oral route was used initially, and we continue to evaluate for other causes of anemia, such as ACD/AI, as well as for other conditions, such as malabsorption. (See "Treatment of vitamin B12 and folate deficiencies" and "Diagnostic approach to anemia in adults".)

Specialist referral — Specialist referral is not generally required for those with suspected vitamin B12 or folate deficiency as the diagnosis is straightforward in the vast majority of cases. However, there may be instances in which specialist referral is indicated, such as the following:

●Concern about other conditions in the differential diagnosis, such as myelodysplastic syndrome, which may require bone marrow evaluation. (See "Diagnostic approach to anemia in adults" and "Approach to the adult with pancytopenia".)

●Concern about gastrointestinal conditions that may require additional evaluations and/or treatments. (See 'Determining the underlying cause of vitamin B12 deficiency' below.)

●Lack of the expected response to therapy. (See "Treatment of vitamin B12 and folate deficiencies".)

DIFFERENTIAL DIAGNOSIS — The differential diagnosis of vitamin B12 and folate deficiencies includes other causes of macrocytic anemia (or macrocytic RBCs without anemia), other causes of pancytopenia or multilobed neutrophils, and other causes of neurologic findings. Many of these findings are quite common in primary care practice. Their full evaluation is discussed in more detail in the linked topic reviews listed for each finding.

●Other causes of macrocytosis and/or anemia with low reticulocyte count – There are numerous other causes of macrocytosis and/or anemia with an impaired reticulocyte response (table 5). Like vitamin B12 and folate deficiency, these other conditions have varied presentations; they often present as an incidental finding or during the evaluation for relatively nonspecific symptoms, such as fatigue. Unlike vitamin B12 and folate deficiency, these conditions have abnormal laboratory findings other than those described above. (See "Macrocytosis/Macrocytic anemia", section on 'Causes of macrocytosis/macrocytic anemia' and "Anemia of chronic disease/anemia of inflammation".)

●Other causes of pancytopenia – Other causes of pancytopenia include a number of bone marrow disorders and autoimmune conditions, some of which can be life-threatening. Like vitamin B12 and folate deficiency, patients with these conditions may present with nonspecific symptoms of cytopenias. Unlike vitamin B12 and folate deficiency, the cytopenias in many of these other conditions can be severe, and there may be a variety of other associated findings. Also unlike vitamin B12 and folate deficiency, these conditions have abnormal laboratory findings other than those described above and diagnosis often requires bone marrow evaluation. (See "Approach to the adult with pancytopenia".)

●Other causes of hypersegmented neutrophils – As noted above, hypersegmented neutrophils are one of the typical findings of megaloblastic anemias, such as vitamin B12 and folate deficiency. Other conditions that can produce hypersegmented neutrophils include other causes of megaloblastic anemia and heat stroke. Like vitamin B12 and folate deficiency, these conditions may be associated with gastrointestinal symptoms and anemia with a low reticulocyte count. Unlike vitamin B12 and folate deficiency, these conditions will have other findings on the examination and laboratory testing. Hypersegmented neutrophils may be seen in individuals treated with hydroxyurea (eg, for sickle cell disease, essential thrombocythemia, or polycythemia vera). (See "Macrocytosis/Macrocytic anemia", section on 'Megaloblastic anemia' and "Evaluation of the peripheral blood smear", section on 'Lobulation'.)

●Other causes of neuropsychiatric findings – Other causes of neurologic findings are numerous. Like vitamin B12 and folate deficiency, some of these conditions are also associated with hematologic abnormalities (eg, copper deficiency, systemic lupus erythematosus, hypothyroidism, hepatic or uremic encephalopathy, infection, medications). Also like vitamin B12 and folate deficiency, findings may be nonspecific, especially in older adults. Unlike vitamin B12 and folate deficiency, these conditions have other abnormal findings on laboratory testing and/or neuroimaging. (See "Evaluation of cognitive impairment and dementia" and "Unipolar depression in adults: Assessment and diagnosis" and "Approach to the patient with sensory loss" and "Overview of cerebellar ataxia in adults" and "Copper deficiency myeloneuropathy".)

●Other causes of fatigue – Fatigue is a nonspecific symptom with numerous causes. Like vitamin B12 and folate deficiency, individuals may have subtle neurologic or neuropsychiatric symptoms. Unlike vitamin B12 and folate deficiency, with these other causes of fatigue, the vitamin B12 and folate levels are normal. Causes and assessment are presented separately. (See "Approach to the adult patient with fatigue".)

POST-DIAGNOSTIC TESTING — Once the diagnosis of vitamin B12 and/or folate deficiency is made, additional historical information and/or testing is used to determine the underlying cause. This is important to prevent future deficiency, to choose the appropriate route and duration of therapy, and to identify other conditions that may require treatment (eg, celiac disease, inflammatory bowel disease [IBD]). Common causes are listed in the tables on causes of vitamin B12 (table 2) and folate (table 3) deficiencies and discussed in more detail separately. (See "Causes and pathophysiology of vitamin B12 and folate deficiencies".)

Other contributing factors to anemia — The diagnosis of vitamin B12 deficiency does not eliminate the possibility of folate deficiency. This is an important consideration in those with folate deficiency, who may have an apparent response to folic acid supplementation with normalization of the hematologic findings and even apparent normalization of the serum vitamin B12 level. Nor does diagnosis of vitamin B12 or folate deficiency eliminate the possibility of other causes of anemia. As an example, many of the conditions that interfere with absorption of vitamin B12 and/or folate also impair the absorption of iron, leading to iron deficiency, and cause inflammation, leading to a component of anemia of chronic disease/anemia of inflammation. (See "Causes and diagnosis of iron deficiency and iron deficiency anemia in adults" and "Anemia of chronic disease/anemia of inflammation".)

Likewise, the diagnosis of folate deficiency does not eliminate the possibility of vitamin B12 deficiency. Individuals with folate deficiency and concomitant vitamin B12 deficiency may have an apparent response to folic acid supplementation with normalization of the hematologic findings and even apparent normalization of the serum vitamin B12 level. However, folic acid cannot correct the neurologic deficits caused by vitamin B12 deficiency; these deficits may become progressive and irreversible if not treated with supplemental vitamin B12. Thus, it is prudent to check the vitamin B12 level in an individual diagnosed with folate deficiency. (See 'Neuropsychiatric changes' above.)

It is also important to assess that treatment has been effective in correcting the deficiency, as discussed separately. (See 'Response to vitamin replacement' above and "Treatment of vitamin B12 and folate deficiencies".)

Determining the underlying cause of vitamin B12 deficiency — The major consideration in an individual with vitamin B12 deficiency involves determining the underlying cause, which in turn determines the route and duration of therapy [21].

As examples, individuals with pernicious anemia (PA; vitamin B12 deficiency due to autoantibodies against intrinsic factor or gastric parietal cells) are often treated with indefinite parenteral vitamin B12 supplementation. However, high-dose oral vitamin B12 (1000 to 2000 mcg daily) is an option if there are no active neurologic complications and the individual is expected to be adherent.

Strict vegans should take an oral supplement, and individuals with celiac disease or IBD may require only temporary oral supplementation until their gastrointestinal disorder is treated/controlled. Treatment will not interfere with the results of this testing and should not be withheld while deciding which testing to pursue.

In a 1994 study, PA was found to account for approximately three-fourths of vitamin B12 deficiency, with treatable intestinal diseases accounting for another 14 percent [70]. It is not clear whether this distribution has shifted along with increasing use of bariatric surgery, use of proton pump inhibitors and metformin, and evolving dietary practices.

Testing for the following may be appropriate, depending on the patient's clinical status, symptoms, and available interventions should an abnormality be found:

●Celiac disease – (See "Diagnosis of celiac disease in children" and "Diagnosis of celiac disease in adults".)

●IBD – (See "Clinical presentation and diagnosis of inflammatory bowel disease in children" and "Endoscopic diagnosis of inflammatory bowel disease in adults".)

●Pancreatic insufficiency – (See "Chronic pancreatitis: Clinical manifestations and diagnosis in adults" and "Exocrine pancreatic insufficiency".)

●Other causes of malabsorption – (See "Chronic complications of short bowel syndrome in children", section on 'Nutritional complications' and "Approach to the adult patient with suspected malabsorption".)

●Hypergastrinemia or gastritis – (See "Physiology of gastrin", section on 'Hypergastrinemia' and "Metaplastic (chronic) atrophic gastritis".)

●Autoimmune thyroiditis – (See "Pathogenesis of Hashimoto's thyroiditis (chronic autoimmune thyroiditis)".)

However, patients should be aware that there are not good data to support this testing.

H. pylori infection has been proposed to be associated with vitamin B12 deficiency, but there is limited evidence of a causal relationship.

In a prospective cohort of 138 patients with anemia and vitamin B12 deficiency who underwent upper gastrointestinal endoscopy, H. pylori was detected in 77 (56 percent) [78]. Of these 77, 31 (40 percent) had improvement in vitamin B12 levels after H. pylori was eradicated. However, as an editorialist noted, the patients were not tested for vitamin B12 absorption or for autoantibodies to IF or gastric parietal cells, and administration of antibiotics might have improved vitamin B12 absorption by treating another cause of deficiency, such as small intestinal bacterial overgrowth [79].

We do not test for H. pylori infection unless the patient has other indications for testing. These are outlined in a separate topic review. (See "Indications and diagnostic tests for Helicobacter pylori infection in adults".)

Additional testing in pernicious anemia — Additional considerations such as screening for gastrointestinal malignancy and the risk of other autoimmune disorders are discussed separately. (See "Treatment of vitamin B12 and folate deficiencies", section on 'Additional considerations for pernicious anemia'.)

SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Anemia in adults".)

INFORMATION FOR PATIENTS — UpToDate offers two types of patient education materials, "The Basics" and "Beyond the Basics." The Basics patient education pieces are written in plain language, at the 5^th to 6^th grade reading level, and they answer the four or five key questions a patient might have about a given condition. These articles are best for patients who want a general overview and who prefer short, easy-to-read materials. Beyond the Basics patient education pieces are longer, more sophisticated, and more detailed. These articles are written at the 10^th to 12^th grade reading level and are best for patients who want in-depth information and are comfortable with some medical jargon.

Here are the patient education articles that are relevant to this topic. We encourage you to print or e-mail these topics to your patients. (You can also locate patient education articles on a variety of subjects by searching on "patient info" and the keyword(s) of interest.)

●Basics topics (see "Patient education: Vitamin B12 deficiency and folate deficiency (The Basics)" and "Patient education: Pernicious anemia (The Basics)")

SUMMARY AND RECOMMENDATIONS

●Prevalence – Vitamin B12 deficiency affects 1 to 2 percent of the general population and 10 to 15 percent of older adults and hospitalized patients. Most cases of vitamin B12 deficiency in resource-rich settings are due to malabsorption rather than dietary deficiency. Folate deficiency is rare in individuals consuming a varied diet with folic acid-supplemented food, as long as they have normal gastrointestinal anatomy and function. (See 'Epidemiology' above.)

●When to suspect – The tables summarize medical conditions and dietary practices associated with vitamin B12 deficiency (table 2) and folate deficiency (table 3). History of using metformin or a proton pump inhibitor, symptoms related to anemia or pancytopenia, gastrointestinal features, neuropsychiatric changes (including irritability or cognitive decline), and neuropathy are also suggestive. In the era of frequent complete blood counts (CBCs), many individuals come to medical attention due to the incidental finding of anemia or macrocytosis. (See 'Clinical presentation' above and 'CBC and blood smear' above.)

●Diagnostic evaluation – The evaluation includes a history for associated conditions and symptoms; an examination for gastrointestinal, dermatologic, neurologic, and other findings; and laboratory testing, including CBC and vitamin B12 and folate levels. We often omit the folate level in those with a folate-enriched diet without gastrointestinal disorders.

The CBC may show macrocytosis, anemia, pancytopenia (typically mild), and/or hypersegmented neutrophils. Laboratory-specific and assay-specific normal values for vitamin B12 and folate should be used if possible (algorithm 1). Bone marrow evaluation is not required. (See 'Overview of evaluation' above.)

•Additional testing (vitamin B12) – Methylmalonic acid (MMA), homocysteine, and/or autoantibodies to intrinsic factor (IF) or gastric parietal cells may be appropriate if the vitamin B12 level is borderline low or discordant with the clinical picture. (See 'Metabolite testing (MMA and homocysteine)' above and 'Autoantibodies to intrinsic factor' above.)

•Additional testing (folate) – MMA, homocysteine, or red blood cell (RBC) folate may be appropriate if the serum folate is borderline or discordant with the clinical picture, especially if the patient has just consumed a folate-fortified meal. (See 'Metabolite testing (MMA and homocysteine)' above and 'RBC folate' above.)

•Response to treatment – Response to administration of the vitamin (especially hematologic response) is supportive. In some cases this may be the sole basis for the diagnosis, although we prefer laboratory testing. (See 'Response to vitamin replacement' above.)

•General approach to evaluating macrocytic anemia – (See "Macrocytosis/Macrocytic anemia".)

●Diagnostic confirmation – A low serum vitamin B12 or folate level is confirmatory, as are increased MMA and homocysteine for vitamin B12 deficiency and homocysteine for folate deficiency (figure 3), with caveats discussed above. Response to vitamin repletion can also be used for diagnostic confirmation. Specialist referral is not required unless there is concern for another hematologic or gastrointestinal condition that should be addressed. (See 'Basis for confirmed diagnosis' above and 'Specialist referral' above.)

●Differential diagnosis – The differential diagnosis includes other causes of macrocytosis, anemia, pancytopenia, hypersegmented neutrophils, neuropsychiatric findings, and fatigue. Many of these findings are common in primary care practice. (See 'Differential diagnosis' above.)

●Determining the cause – Other testing may be appropriate to determine the cause, and route and duration of therapy. Individuals with pernicious anemia may require increased monitoring for gastrointestinal malignancy, those with celiac disease may require additional attention to bone health and other nutritional deficiencies, and most individuals with folate deficiency require testing of vitamin B12 levels before folic acid is administered. (See 'Post-diagnostic testing' above and "Causes and pathophysiology of vitamin B12 and folate deficiencies".)

●Management – (See "Treatment of vitamin B12 and folate deficiencies".)

ACKNOWLEDGMENTS

UpToDate gratefully acknowledges Stanley L Schrier, MD (deceased), who contributed as Section Editor on earlier versions of this topic and was a founding Editor-in-Chief for UpToDate in Hematology.

The UpToDate editorial staff also acknowledges the extensive contributions of William C Mentzer, MD, to earlier versions of this and many other topic reviews.