Megaloblastic anemia

What every physician needs to know about megaloblastic anemia?

Megaloblastic anemias arise from a common defect in DNA synthesis that leads to a distinctive morphological pattern within rapidly proliferating cells within the bone marrow.

While commonly induced by anti-cancer or anti-viral drugs, megaloblastic anemia has been traditionally ascribed to a deficiency of either vitamin B12 (also known as cobalamin) or folate, which normally function as coenzymes for important reactions that lead to the synthesis of three of the four nucleotides of DNA. Since substantial DNA synthesis is required by rapidly dividing hematopoietic cells, when there is either folate or vitamin B12 deficiency, these cells are left in limbo with DNA values between 2N and 4N as they continue in vain, to double their DNA prior to cell division.

Megaloblastic cells have a characteristic morphology consisting of an immature nucleus with finely particulate chromatin within a larger than normal cell, which has a cytoplasm that otherwise appears normal. This nuclear-cytoplasmic asynchrony is most obvious in orthochromatic (hemoglobinized) normoblasts, or giant myelocytes and metamyelocytes. Although there is profound ineffective erythropoiesis with intramedullary hemolysis, the presence of macroovalocytes and hypersegmented polymorphonuclear neutrophils in the peripheral smear are reliable clues that point back to a megaloblastic marrow.

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The recognition of megaloblastic anemia should be followed by efforts to distinguish whether folate, vitamin B12, or combined folate and vitamin B12 deficiencies are the cause of the anemia; then to identify the underlying disease and etio-pathophysiological mechanism that led to the deficiency in the first place. This will help you decide the type of vitamin replacement, the dose, and the duration of treatment.

Anemia due to megaloblastic anemia is usually reticulocytopenic. Distinguishing between macrocytes and macroovalocytes on a blood smear is important, because it is the macroovalocytes (which have a central pallor that occupies less than one-third the diameter of a large cell, with a mean corpuscular volume usually over 105fL) that are usually associated with megaloblastic anemia. Macrocytes, on the other hand (which have a central pallor that occupies more than one-third of the diameter of the red cell), are actually thin macrocytes, and can be found in several non-hematologic conditions. These conditions include the post-splenectomy state, liver disease, alcoholism, myelophthisic or hypoplastic anemias, hypothyroidism, chronic lung disease, smoking, hyperglycemia, and even leukocytosis.

What features of the presentation will guide me toward possible causes and next treatment steps:

Anemia with macroovalocytes and hypersegmented polymorphonuclear leukocytes found in a patient with mild neutropenia and mild thrombocytopenia may be initial presentations. Alternatively, an obvious primary disease that is associated with folate deficiency anemia may dominate the clinical picture (for example, alcoholism, malabsorption, or malnutrition). Vitamin B12 deficiency could present primarily with dominant neurologic and/or psychiatric disease associated with milder anemia (which is the usual presentation in the United States of America [USA]). Often, there is an inverse correlation between the severity of neurologic symptoms and the severity of anemia in vitamin B12 deficiency.

Other presentations include cardiopulmonary symptoms attributable to anemia, thrombosis due to chronic hyperhomocysteinemia, hyperpigmentation of the skin, premature graying, infertility, or neuropsychiatric presentations with subacute combined degeneration of the spinal cord.

Clues to the diagnosis from the history that can help point to either folate or vitamin B12 deficiency:
  • Dietary history associations

– Vegetarianism/food faddism – vitamin B12 deficiency.

  • Medical history associations

– Hemoglobinopathies (hemolytic anemias) – folate deficiency

– Celiac disease – folate plus iron deficiency

– Epilepsy on anticonvulsants – folate deficiency

– Autoimmune diseases (pernicious anemia) – vitamin B12 and iron deficiency

  • Travel history associations

– Tropical sprue/malaria – folate deficiency.

  • Surgical history associations

– Gastrectomy – vitamin B12 and iron deficiency

– Ileal resection/radiation – vitamin B12 deficiency

  • Occupational/recreational associations

– Dentist, anesthesiologist, or personnel: nitrous oxide (N20) – vitamin B12 deficiency.

The underlying condition predisposing to the development of folate deficiency usually will have begun within the previous 6 months and often dominates the overall clinical picture. By contrast, vitamin B12 deficiency takes several years to manifest clinically and can be insidious, especially when there are no other problems related to malabsorption.

Physical examination associations:
  • If patient is well nourished, think of vitamin B12 deficiency (vegetarian/pernicious anemia)

  • If patient is poorly nourished, with additional deficiency, think of malabsorption and folate deficiency (with deficiency of vitamins A, D, and K or protein-calorie malnutrition, or both) when the patient has a flat affect, is elderly, or has psychiatric illness

  • Anemia can result in varying degrees of pallor and mild jaundice (which reflects intramedullary hemolysis of megaloblastic cells)

  • Angular cheilosis, glossitis with a smooth (depapillated), beefy-red tongue

  • Anemia can result in heart failure

  • Diffuse brownish pigmentation, abnormal blotchy tanning, or vitiligo and premature graying (vitamin B12 deficiency)

  • Look for other features of surgical scars, autoimmune disease

  • Neurologic abnormalities suggest vitamin B12 deficiency with varying degrees of involvement of the posterior columns, as well as the pyramidal, spinocerebellar, and spinothalamic tracts

  • With profound megaloblastic anemia from folate or vitamin B12 deficiency, there can be mild fever, hepatosplenomegaly, with pancytopenia and petechial hemorrhages. If there are neurologic manifestations, think of vitamin B12 deficiency. In the USA, neurologic features may be dominant with only minimal anemia.

What laboratory studies should you order to help make the diagnosis and how should you interpret the results?

Evaluation of the blood, that is, complete blood count, peripheral smear, basic blood chemistry

Anemia (can be as low as 5gm/dl) with reticulocytopenia can be associated with a serial rise in mean corpuscular volume over several months or years and mild neutropenia (rarely less than 1,000 per microL) and thrombocytopenia (rarely less than 50,000 per microL) can be seen.

The next step is to look at the peripheral smear to differentiate between thin macrocytes and macroovalocytes (up to 14µm) and also identify if there are over 5% hypersegmented (5-lobed) polymorphonuclear leukocytes. There may also be occasional tear-drop cells with reduction in platelets seen on the smear. If nucleated red cells with features of orthochromatic megaloblasts are observed on the smear, this would be an accurate reflection of megaloblastic hematopoiesis in the bone marrow and help to clinch the diagnosis. These orthochromatic megaloblasts, which contain hemoglobin in the cytoplasm also retain a large, finely stippled, sieve-like immature nucleus; this is in sharp contrast to the clumped chromatin of orthochromatic normoblasts of a similar stage.

Ineffective erythropoiesis associated with intramedullary hemolysis will be picked up by low haptoglobin, increased lactate dehydrogenase (LDH) up to 1,000 units, a mild increase in bilirubin, and occasional red cell fragments seen on the blood smear.

The next cost-effective tests to obtain in patients suspected of having megaloblastic anemia are serum vitamin B12 and serum folate levels. Note that these tests are valuable primarily when there is preexisting anemia or neuropsychiatric disease that is, when the pre-test probability is high.

  • Low serum vitamin B12 levels

If there is megaloblastic anemia or neurologic or psychiatric disease that you suspect is due to vitamin B12 deficiency, the finding of low serum vitamin B12 levels (less than 200pg/ml) will support this diagnosis; if the vitamin B12 is between 200 and 300pg/ml and you still suspect this diagnosis, then you should pursue additional tests to confirm if both metabolites (serum methylmalonic acid and homocysteine are increased – this is further discussed below).

  • Abnormal blood counts, serum vitamin B12 is over 300pg/ml, but serum folate level is less than 2ng/ml

If you suspect megaloblastic anemia on the basis of abnormal blood counts and the serum vitamin B12 is over 300pg/ml but the serum folate level is less than 2ng/ml, this will be consistent with folate deficiency; (note: if the serum folate level is between 2 and 4ng/ml and you still suspect folate deficiency, this is when you pursue additional tests to evaluate if the serum homocysteine is increased to confirm the diagnosis; this is discussed further below).

  • Serum vitamin B12 is less than 200pg/ml and the serum folate level is less than 2ng/ml

If the serum vitamin B12 is less than 200pg/ml and the serum folate level is less than 2ng/ml, this is consistent with a combined vitamin B12 and folate deficiency.

  • Serum vitamin B12 is over 300pg/ml and the serum folate is over 4ng/ml

If the serum vitamin B12 is over 300pg/ml and the serum folate is over 4ng/ml, then both vitamin B12 and folate deficiency are unlikely, so the anemia is likely due to another cause.

A clinical pearl to remember

In countries where food is not fortified with folic acid, if there is a blood picture consistent with megaloblastic anemia with or without neurologic-psychiatric findings, and the serum vitamin B12 is less than 200pg/ml, but the serum folate is normal (over 4ng/ml), although these findings are consistent with vitamin B12 deficiency, there is still the possibility of an associated (but masked) folate deficiency. This is because folate cannot be utilized by vitamin B12 deficient cells and only upon treatment with vitamin B12 can the folate will be utilized. When this occurs, the underlying folate deficiency can become manifested with serum folate dropping to a frankly deficient level of less than 2ng/ml, and unless folate is given at this juncture, there will continue to be a suboptimal response of the anemia to vitamin B12 alone.

As noted above, if there are clinical findings suggestive of vitamin B12 and/or folate deficiency but results are not below normal (that is, the serum vitamin B12 is between 200 and 300pg/ml or the serum folate is between 2 and 4ng/ml), you will need to pursue additional metabolite tests like serum homocysteine and methylmalonic acid to help confirm the diagnosis. These are discussed below, under ‘When do you need to get more aggressive tests’.

Remember that falsely low serum vitamin B12 concentrations can also be seen in one-third of those with folate deficiency, and in other conditions (pregnancy, multiple myeloma, or in anyone consuming megadose vitamin C therapy).

A more dangerous situation occurs when there are falsely high vitamin B12 levels in the face of a true vitamin B12 deficiency. This can occur when vitamin B12 is released from liver stores in acute hepatic necrosis, or when vitamin B12-binding proteins are released by activated macrophages (during autoimmune diseases, monoblastic leukemias, lymphomas, myeloproliferative disorders, or with hepatic tumors).

The serum folate is highly labile and a patient with a true reduction in serum folate on admission can have a falsely elevated level following a single nutritious hospital meal.

Anticonvulsants can prevent folate absorption from the small intestine. Alcoholism poses special problems since one-third to one-half of such patients with true folate deficiency may have a normal or borderline low level, so one approach to reduce ambiguity, is to assume they are deficient and treat empirically with folate.

What conditions can underlie megaloblastic anemia:

Vitamin B12 deficiency is for the most part either due to either dietary insufficiency from vegetarianism or near-vegetarianism (when animal source foods are consumed infrequently by non-vegetarians) or due to malabsorption from any variety of causes. The breast fed infant of an undiagnosed vitamin B12 deficient mother is at risk of severe manifestations.

If the consumption of vitamin B12 is curtailed abruptly, because only 1mcg is lost every day, the body’s stores of between 2 to 5mg of vitamin B12 can last for up to 5 to 10 years. However, any interruption of the enterohepatic pathway, which normally turns over 5 to 10mcg daily can manifest a deficiency much earlier, especially when vitamin B12 stores are low to begin with.

All vegetarians (in either developing or developed countries) who do not routinely take supplements of vitamin B12 are at risk for developing vitamin B12 deficiency. In addition, non-vegetarians who infrequently eat animal source foods (that are normally rich in vitamin B12) are at risk for deficiency.

Vitamin B12 malabsorption can arise due to several causes in the gastrointestinal tract. In the stomach, achlorhydria leads to inadequate dissociation of vitamin B12 from food protein, leading to food-vitamin B12 malabsorption. A deficiency of intrinsic factor (IF) can arise from either gastrectomy or following the autoimmune destruction of IF producing cells (pernicious anemia). A failure to effect a normal transfer of vitamin B12 bound to R-protein to IF as a result of inadequate pancreatic protease can also result in malabsorption. Vitamin B12 may be usurped by excess intestinal bacterial overgrowth or by worms before it reaches ileal IF-vitamin B12 receptors. Moreover, loss of these receptors from either disease or loss of 1 to 2 feet of terminal ileum can also interfere with absorption. Finally, interference with the integrity of the vitamin B12 molecule by inhaled nitrous oxide can also lead to a functional B12 deficiency.

Folate deficiency is commonly due to decreased dietary intake. In the USA, folate deficiency has nearly been completely eliminated by fortification of flour with folic acid. However, young women who eat low carbohydrate diets, as well as socially isolated individuals, or those with chronic intestinal disease (tropical sprue) with anorexia may continue to be at risk for folate deficiency. Increased requirements (short interpregnancy intervals) is another common cause and when coupled with poor intake (hyperemesis gravidarum) this can lead to devastating outcomes for the newborn of mothers with folate deficiency. This is more commonly seen in developing countries.

Folate is also required during periods of intense hematopoiesis (myeloproliferative disease) or during compensatory hematopoiesis with chronic hemolysis of any cause. If folate levels are not replenished, the patient can develop an acute reticulocytopenic crisis with shut-down of hematopoiesis. Therefore, based on the clinical setting that gives rise to megaloblastic anemia, the cause of the folate and vitamin B12 deficiency should be revealed through a careful history and physical examination.

When do you need to get more aggressive tests:

A bone marrow aspirate would provide results (within an hour) to confirm whether or not there was a megaloblastic bone marrow in a patient with moderately severe anemia. However, if there is a primary neurologic presentation, or there is mild to moderate anemia and there is little urgency to make the diagnosis, one can elect to initiate the work-up with serum folate or serum vitamin B12 levels and more specialized metabolite studies.

The following is a stepwise guide to laboratory testing when the results from serum vitamin B12 and serum folate are equivocal and more specialized testing using serum metabolites (homocysteine and methylmalonic acid levels) are required.

General considerations with metabolites

In the event that the initial serum vitamin B12 or serum folate test results give equivocal results because they are in the low-to-normal range, additional testing of serum methylmalonic acid and homocysteine levels is useful because normal metabolite levels rule out folate and vitamin B12 deficiency.

In vitamin B12 deficiency, both these metabolites tend to rise in proportion to the severity of deficiency with an elevation of serum methylmalonic acid levels (normal is less than 270nM) and serum homocysteine (normal is less than 14 microM).

In the USA, the median value for serum methylmalonic acid levels in patients with clinically confirmed vitamin B12 deficiency is 3,500nM and the median homocysteine value is 70 microM.

In the case of folate deficiency, there will only be a rise in homocysteine (with normal serum methylmalonic acid levels). In folate deficiency, the median value of serum homocysteine is 50 microM.

Specific scenarios for interpretation

If there is a suspicion of megaloblastic anemia and/or neurologic-psychiatric disease consistent with vitamin B12 deficiency, but the serum vitamin B12 is between 200 and 300pg/ml and the serum folate is over 4ng/ml, an elevation of serum methylmalonic acid and homocysteine would point to the diagnosis of vitamin B12 deficiency.

If the serum vitamin B12 is less than 200pg/ml and the serum folate is also less than 2ng/ml, this finding would be consistent with a combined vitamin B12 and folate deficiency. However, remember that one-third of patients with folate deficiency can also have low vitamin B12 levels (and this could be the case in this scenario). So here, the added information from a high methylmalonic acid will confirm the diagnosis of a combined folate and vitamin B12 deficiency. In resource poor settings, an alternative to obtaining the more expensive metabolite levels would be to simply redraw vitamin B12 levels after treatment of folate deficiency; if the levels of vitamin B12 are still low, this would retrospectively point to an underlying vitamin B12 deficiency and trigger the need for therapy with vitamin B12 replacement also for this patient.

If there is a suspicion of megaloblastic anemia and the serum vitamin B12 is over 300pg/ml and the serum folate is between 2 and 4ng/ml, then the finding of an increase in serum homocysteine would support the diagnosis of folate deficiency. If however, the serum homocysteine is normal, this would suggest a megaloblastoid process that is not related to vitamin deficiency. The differential diagnosis would then center on an effect of chemotherapy, an immunosuppressive or antiretroviral drug, or a rare manifestation of an intrinsic hematologic disease like erythroleukemia or myelodysplastic syndrome (especially the 5 q- syndrome).

Important caveats when using metabolite levels:
  • Both homocysteine and methylmalonic acid levels rise with dehydration or renal failure

  • Correct replacement will reduce the level of metabolites to normal in a week

  • Conditions mentioned above that are known to falsely elevate serum folate or vitamin B12 levels

Other scenarios where results of elevated serum methylmalonic acid and/or homocysteine can help to clarify ambiguous presentations include conditions mentioned above that are known to falsely elevate serum folate or vitamin B12 levels. In this scenario, an elevation of both of these metabolites can clarify the diagnosis of true vitamin B12 deficiency (but still cannot rule out an combined vitamin B12 plus folate deficiency). However, in pure folate deficiency, the methylmalonic acid will be normal but homocysteine elevated. When both values are normal, this would rule out deficiency of both.

  • Methylmalonic acid remains elevated after treatment with vitamin B12

If methylmalonic acid remains elevated after treatment with vitamin B12 (and homocysteine is also normal) this could suggest overgrowth of intestinal bacteria as the cause of elevated methylmalonic acid. Treatment with a short course of broad spectrum antibiotics usually reduces the serum methylmalonic acid level in this scenario.

  • Finally, recall that the elevation of both metabolites cannot rule out a combined vitamin B12 and folate deficiency

  • Isolated increase in serum methylmalonic acid and/or homocysteine values without clinical findings suggests a subclinical deficiency

The only way one can confidently make the diagnosis of subclinical vitamin B12 and/or folate deficiency based purely on increased serum methylmalonic acid and/or homocysteine values, is to show that full dose therapy with vitamin B12/folate led to a reduction in these elevated metabolites.

What imaging studies (if any) will be helpful?

Although all causes of vitamin B12 malabsorption can be managed the same way (with either monthly vitamin B12 injections or high-dose daily oral vitamin B12), the value in identifying the etiology would be to remove the offending cause, provided this is a simple matter.

Because of the current non-availability of the Schilling test in the USA (since 2003), you will need to rely on a good history and physical examination, and judiciously use other tests to help identify the cause of vitamin B12 malabsorption.

The key point here is that many of these diagnoses can be made through a detailed history (gastric bypass or gastrectomy), physical examination (abdominal scars), and additional specific tests. These include elevated serum gastrin in Zollinger-Ellison syndrome, or elevated serum anti-tissue transglutaminase antibodies in celiac disease. Identification of serum anti-intrinsic factor antibodies (found in 60% with pernicious anemia) would clinch the diagnosis and mandate lifelong replacement with vitamin B12.

Consultation with a gastroenterologist could help identify other causes of malabsorption (stasis, strictures, or fistulas), or lead to diagnosing ova in the stool and lead to elimination of Diphyllobothrium latum worms with a single dose of an antihelminthic, or diagnosis and treatment of bacterial overgrowth in the clinical setting of either scleroderma, blind pouches, or fistulas from Crohn’s disease with metronidazole, or in managing celiac disease with a gluten-free diet, or in treating tropical sprue with folate and a tetracycline.

For children and adolescents with megaloblastic anemia, juvenile pernicious anemia can be distinguished from congenital intrinsic factor deficiency by the finding of achlorhydria. Mutations in IF, or the ileal IF-vitamin B12 receptor, which is composed of two genes (amnionless, cubulin genes), can identify hereditary megaloblastic anemia and Imerslund-Gräsbeck syndrome, but again, irrespective of the cause, these children will require lifelong vitamin B12.

What therapies should you initiate immediately and under what circumstances – even if root cause is unidentified?

A patient who is acutely decompensated with heart failure from anemia should be transfused with a single unit of packed red cells under aggressive diuretic coverage. At this point, some experts would give 1mg of vitamin B12 and 1mg of folic acid together parenterally (but only after all laboratory studies have been drawn, including tests for anti-intrinsic factor assays when pernicious anemia is suspected).

Once a diagnosis of vitamin B12 deficiency is confirmed, an aggressive protocol to replace vitamin B12 is 1mg/day of intramuscular or subcutaneous vitamin B12 (cyanocobalamin) (week 1), 1mg twice weekly (week 2), 1mg/week for 4 weeks, and then 1mg/month can be given. This regimen will replenish vitamin B12. The duration of therapy is dictated by the likely underlying cause of the deficiency. In cases of pernicious anemia and food vitamin B12 malabsorption, this will be for the rest of the life of the patient. Alternatively after acute replacement over a month is completed, the patient may be switched to oral vitamin B12 tablets (1mg) daily.

Even if there is malabsorption, about 1 to 2% of an oral dose is absorbed by passive diffusion; this allows for absorption of 10 to 20 micrograms of vitamin B12, when 1 to 2 mg of vitamin B12 are consumed daily. This amount is more than sufficient to meet daily requirements.

A less aggressive regimen would be to treat those with minimal symptoms or signs of vitamin B12 deficiency primarily with oral vitamin B12 at a dose of 2mg/day. Those who refuse injections or have bleeding problems can also be treated with oral vitamin B12.

For vegetarians who do not malabsorb food vitamin B12, much smaller doses of 5 to 10 micrograms of vitamin B12 replacement will suffice as maintenance therapy throughout life; however if a vegetarian is diagnosed with megaloblastic anemia, because unreplenished stores can lead to neurologic dysfunction, prudence dictates that the depleted stores must first be replenished preferably with parenteral vitamin B12 before moving to maintenance therapy. If there is resistance to injections, however, treat such patients initially with 2mg per day of oral vitamin B12 for the first 3 months and then use the lower maintenance dose in the long term.

In the case of folate deficiency, oral folate (folic acid) should be given, at doses of 1 to 5mg/day results in adequate absorption (even if the intestine is diseased) and therapy continued until there is complete hematologic recovery. Folinic acid should be reserved for megaloblastic anemia that is induced by methotrexate, trimethoprim-sulfamethoxazole, or due to nitrous oxide inhalation.

Remember that current levels of food fortification with folic acid are not likely to mask any vitamin B12 related neurologic disease if the patient is not taking additional supplements of folate.

What other therapies are helpful for reducing complications?

For vegetarians, prophylaxis with vitamin B12 is indicated

Use 5 to 10 micrograms per day (in the form of vitamin B12-fortified foods or oral vitamin B12 tablets (which are the cheapest). For seniors and those with any other causes that predict for food vitamin B12 malabsorption (see Pathophysiology) treatment with vitamin B12 (1 to 2mg) tablets taken orally each day is appropriate; smaller doses are inadequately absorbed for this group.

Prophylaxis with folic acid
  • Women of childbearing age

All women of childbearing age should receive 400 micrograms of folic acid a day periconceptionally and throughout pregnancy and lactation (that is, all the time). However, those with prior delivery of a baby with a neural-tube defect should be given 4mg per day to prevent a recurrence of neural-tube defects.

  • Patients taking anticonvulsant and immunosuppressive medications

All patients taking anticonvulsant medications (phenytoin, phenobarbital, carbamazepine) should take 1mg folic acid per day, as should those patients on methotrexate for immunosuppression (rheumatoid arthritis, psoriasis).

  • Patients with myeloproliferative diseases or chronic hemolysis

Patients with myeloproliferative diseases or chronic hemolysis from any cause, including those with malaria who are treated with anti-malarials, should be given prophylaxis with 1mg folic acid daily. Remember that use of higher doses of folic acid can interfere with the effectiveness of anti-folate anti-malarials.

What should you tell the patient and the family about prognosis?

The vast majority of patients with vitamin B12 deficiency will respond to vitamin B12 replacement with a dramatically improved sense of well being. The reinitiation of normal hematopoiesis within a day will manifest as a peak in reticulocyte count between days 5 to 8, and eventual normalization of counts by 2 months. While most mild neurologic abnormalities that may have arisen in the past 3 months can be expected to improve in up to 90% of patients within about 6 months, those with more prolonged symptoms could take a year to recover completely. Persistence of neurologic dysfunction beyond a year portends potential irreversibility.

If there is no improvement in megaloblastic hematopoiesis, the differential diagnosis should include associated hypothyroidism, iron deficiency (common in pernicious anemia/gastrectomy), or the presence of a hemoglobinopathy, or primary bone marrow disorders, and treatment should be adjusted accordingly.

“What if” scenarios.

Looking solely at the mean corpuscular volume in lieu of looking at the peripheral smear can be misleading. For example, if macroovalocytes from vitamin B12 or folate deficiency are also associated with microcytic cells from an associated iron deficiency or thalassemia, the mean corpuscular volume could be normal; in this scenario, the red cell distribution width would be large.

The additional finding of hypersegmented polymorphonuclear neutrophils should point to the likelihood of megaloblastic anemia, and a bone marrow aspirate could clinch the diagnosis when giant myelocytes and metamyelocytes are seen (even though abnormal megaloblastic erythroblasts are not seen because of a hemoglobinization defect in iron deficiency or a thalassemia). Severe fragmentation and high LDH (with low haptoglobin and mild bilirubinemia) may lead to a misdiagnosis of thrombotic microangiopathy. This will be picked up by looking at a peripheral smear.

If iron is given alone to a patient with combined iron plus vitamin B12 and/or folate deficiency, the megaloblastic features will become unmasked and fully manifest with macroovalocytes and hypersegmented polymorphonuclear neutrophils. Moreover, if there is combined iron plus folate and/or vitamin B12 deficiency, replacing either one without the other will fail to fully correct the anemia. Likewise, associated hypothyroidism will preclude a maximal hematologic response to vitamin B12 plus iron in pernicious anemia.

Seniors with vitamin B12 deficiency may have anemia of chronic renal failure and poor response to vitamins.

In resource poor settings with chronically poor vitamin B12 intake (due to low consumption of animal source foods) as well as a poor daily folate intake, a low serum vitamin B12 may be accompanied by a normal serum folate level (because the folate is not utilized by cells during vitamin B12 deficiency). Subsequent treatment of such a patient with vitamin B12 alone may then result in a fall in serum folate levels to frankly deficient values, and only replacement with folic acid will help to restore hemoglobin values to optimum. A simple practical approach is to empirically treat all those with vitamin B12 deficiency with both vitamin B12 and folate to replenish stores, so as to not undertreat those with masked folate deficiency.

Conversely, in such resource poor settings, if both the serum folate and vitamin B12 are low to begin with, this could well be due to a combined vitamin deficiency that again warrants giving both vitamins.

However, because a low serum folate can also lead to a falsely low vitamin B12 level (and obtaining a serum methylmalonic acid level to help decipher these possibilities may be impossible) you could elect to treat the patient with folic acid (1mg daily) alone for a couple of weeks, and then re-draw the serum vitamin B12 to see if this level has normalized. If it has not normalized, then you will conclude that this patient actually had vitamin B12 deficiency that led to combined low serum folate and vitamin B12 levels in the first place.

Of course, the default and minimalist approach in resource poor settings (where the cost of repeating testing could be prohibitive for the patient) would be to empirically treat all patients who are found to have a low serum folate (and/or) vitamin B12 levels as though they have a combined nutritional folate and vitamin B12 deficiency (with 1mg folic acid and 10 microgram vitamin B12 daily). If there is an incomplete response in the hemoglobin values, then moving to a higher oral vitamin B12 dose of 1mg daily would be indicated to optimize the response in such a patient with malabsorption of vitamin B12. However, remember that in some countries, there may be minimal cost differences between 10 microgram and 1mg of vitamin B12 tablets (in which case the higher dose may be preferable up front in this scenario).

Alcoholism, which can primarily lead to folate deficiency, can also result in a low vitamin B12 level in one-third of patients. In an acute presentation of such an individual, the presence of associated thiamine deficiency can result in peripheral neuropathy with dry beriberi and Wernicke-Korsakoff syndrome, but could also raise the possibility of vitamin B12 deficiency. With the (often retrospective) receipt of a low vitamin B12 level a week later, which is a falsely low result (from pure folate deficiency), a wrong attribution of the presentation to vitamin B12 deficiency can be avoided by obtaining a methylmalonic acid level, which would only be high if there was true vitamin B12 deficiency (warranting long term vitamin B12 replacement).


Etio-pathophysiologic classification of vitamin B12 deficiency

Nutritional vitamin B12 deficiency (insufficient intake of vitamin B12-rich animal source foods)

Vegetarians, near-vegetarians, breast-fed infants of mothers with pernicious anemia or other causes of vitamin B12 malabsorption.

Abnormal intragastric events (inadequate proteolysis of food bound vitamin B12)

Atrophic gastritis, partial gastritis with hypochlorhydria, proton-pump inhibitors, H2 blockers, gastric bypass.

Loss/atrophy of gastric oxyntic mucosa (deficient intrinsic factor [IF] molecules)

Total or partial gastrectomy, adult and juvenile pernicious anemia, caustic destruction (lye)

Abnormal events in the small bowel lumen

  • Inadequate pancreatic protease (R factor-vitamin B12 complex is not degraded, so vitamin B12 is not transferred to IF)

– Insufficient pancreatic protease (pancreatic insufficiency)

– Inactivation of pancreatic protease (Zollinger-Ellison syndrome)

  • Usurping of luminal cobalamin (inadequate binding of cobalamin to IF)

Disorders of ileal mucosa/IF, cobalamin receptors (IF-vitamin B12 complex is not bound to ileal IF-vitamin B12 receptors)

  • By bacteria in stasis syndromes (blind loops, pouches of diverticulosis, strictures, fistulas, anastomosis), scleroderma, or hypogammaglobulinemia

  • By D. latum (fish tapeworm)

  • Diminished or absent IF-vitamin B12 receptors: Ileal bypass/resection/fistula

  • Abnormal mucosal architecture/function: Tropical/non-tropical sprue, Crohn’s disease

  • IF-vitamin B12 receptor defects: Immerslund-Gräsbeck syndrome, hereditary megaloblastic anemia (rare)

  • Drug-effects: Metformin, cholestyramine, colchicine, neomycin

Congenital disorders of plasma vitamin B12 transport (transcobalamin II [TC11] is missing or TCII bound vitamin B12 fails to bind to TCII receptors) (rare)

Metabolic disorders (missing/abnormal enzymes required to process vitamin B12 into coenzyme forms within cells)

  • Congenital: Several enzymes are missing or are mutated (rare)

  • Acquired disorders (vitamin B12 molecule is functionally inactivated by irreversible oxidation): Nitrous oxide inhalation (occupational exposure or through recreational use)

Etio-pathophysiologic classification of folate deficiency

Nutritional causes

  • Decreased dietary intake(folate-rich foods not consumed)

– Poverty and famine, institutionalized individuals (psychiatric or nursing homes)

– Anorexia from chronic debilitating disease

– Special diets or food fads

– Use of cultural/ethnic cooking techniques that destroy food folate

  • Decreased diet plus increased requirements

– Physiologic: Pregnancy and lactation, prematurity, hyperemesis gravidarum, infancy

– Pathologic: Intrinsic hematologic diseases (hemolysis with compensatory erythropoiesis or excess hematopoiesis in myeloproliferative states) or dermatologic disease (psoriasis)

  • Folate malabsorption

  • Folate malabsorption with mucosal abnormalities

Tropical and nontropical sprue, regional enteritis.

  • Folate malabsorption with normal intestinal mucosa

– Hereditary folate malabsorption (mutations in proton-coupled folate transporters) (rare)

– Drugs: Sulfasalazine, pyrimethamine, proton-pump inhibitors (inhibition of proton-coupled folate transporters)

Defective CSF folate transport (rare)

  • Hereditary folate malabsorption (mutations in proton-coupled folate transporters in intestine and choroid plexus)

  • Acquired: Cerebral folate deficiency (auto-antibodies to folate receptors triggered by consuming cow’s milk)

Inadequate cellular utilization of folates

  • Folate antagonists (methotrexate)

  • Hereditary enzyme deficiencies involving folate (rare)

Drugs (via multiple effects on folate metabolism)

Alcohol, sulfasalazine, pyrimethamine, trimethoprim-sulfamethoxazole, diphenylhydantoin, barbiturates, niacin

Miscellaneous megaloblastic anemias (unrelated to either vitamin B12 or folate deficiency)

Congenital disorders of DNA synthesis (very rare)

Orotic aciduria, Lesch-Nyhan syndrome, congenital dyserythropoietic anemia, and thiamine responsive megaloblastic anemia.

Acquired disorders of DNA synthesis

  • Hematologic diseases: erythroleukemia, refractory sideroblastic anemias

  • All antineoplastic and immunosuppressive drugs that inhibit DNA synthesis

  • Antinucleosides used against HIV and other viruses

  • Direct toxic effects on the bone marrow by alcohol

What other clinical manifestations may help me to diagnose megaloblastic anemia?

For folate deficiency, think of patients who are infirm, chronically sick, alcoholic, women with short pregnancy intervals, those on a low carbohydrate diet, a patient with a psychiatric disease whose diet is unsupervised, or someone on a food fad.

Because 30% of patients with pernicious anemia have a family history, they could develop additional autoimmune diseases, especially endocrinopathies.

New clinical presentations to think about underlying vitamin B12 as the cause include adverse pregnancy outcomes (preeclampsia, placental abruption or infarction, recurrent miscarriage) and poor pregnancy outcomes (preterm delivery, neural tube defects, congenital heart defects, intrauterine growth retardation), very early recurrent miscarriage, and reduced neurocognitive performance in children.

Those with incidental diagnosis of hyperhomocysteinemia should also be screened for folate/vitamin B12 deficiency. Hyperhomocysteinemia is also associated with reduced bone mineral density and increased risk for hip fracture, small vessel cerebrovascular disease-related stroke, and age-related macular degeneration. Reducing high serum homocysteine levels with folate/vitamin B12/pyridoxine can prevent these complications.

Because folate supplements (in combination with vitamin B12 in some studies) can help reduce the rate of cognitive decline among healthy elderly patients, reduce age related (sensorineural) hearing loss, and age-related macular degeneration, elderly patients presenting with such problems should be evaluated for potentially having chronic folate or vitamin B12 deficiency.

The well-known effect of periconceptional folate supplements to reduce first occurrence and as well as the recurrence of neural tube defects in subsequent births is well documented.

The benefits of folate fortification of food has also been corroborated in population-based studies and it is also thought that additional midline birth defects are also likely to be reduced. Hence, in populations where food folate fortification is not practiced, such midline birth defects should also raise questions of possible longstanding folate deficiency.

What other additional laboratory studies may be ordered?


What’s the evidence?

Carmel, R. “How I treat cobalamin (vitamin B12) deficiency”. . vol. 112. 2008. pp. 2214-2221. [A succinct contemporary approach to vitamin B12 deficiency]

Kuzminski, AM, Del Giacco, EJ, Allen, RH, Stabler, SP, Lindenbaum, J. “Effective treatment of cobalamin deficiency with oral cobalamin”. . vol. 92. 1998. pp. 1191-1198. [Shows that oral vitamin B12 at 2mg per day is a sufficient substitute to maintenance monthly subcutaneously vitamin B12 shots]

Sato, Y, Honda, Y, Iwamoto, J. “Effect of folate and mecobalamin on hip fractures in patients with stroke: a randomized controlled trial”. . vol. 293. 2005. pp. 1082-1088. [Demonstration of benefits of vitamin B12 and folate supplementation on bone strength]

Hodis, HN, Mack, WJ, Dustin, L. “High-dose B vitamin supplementation and progression of subclinical atherosclerosis: a randomized controlled trial”. . vol. 40. 2009. pp. 730-736. [Shows benefits of B-vitamins in reducing subclinical atherosclerosis – this is the likely basis for primary prevention of stroke]

Christen, WG, Glynn, RJ, Chew, EY. “Folic acid, pyridoxine, and cyanocobalamin combination treatment and age-related macular degeneration in women: the Women’s Antioxidant and Folic Acid Cardiovascular Study”. . vol. 169. 2009. pp. 335-341. [Demonstrates benefits of supplementation of folate, vitamin B12, and vitamin B6, to prevent age-related macular degeneration in women]

Durga, J, van Boxtel, MP, Schouten, EG. “Effect of 3 year folic acid supplementation on cognitive function in older adults in the FACIT trial: a randomised, double blind, controlled trial”. . vol. 369. 2007. pp. 208-216. [Shows benefits of folate supplementation which led to improved cognitive function]

Durga, J, Verhoef, P, Anteunis, LJ. “Effects of folic acid supplementation on hearing in older adults: a randomized, controlled trial”. . vol. 146. 2007. pp. 1-9. [Shows benefits of folate supplementation that led to reduced sensorineural deafness]

Bukowski, R, Malone, FD, Porter, FT. “Preconceptional folate supplementation and the risk of spontaneous preterm birth: a cohort study”. . [Identifies the benefits of folate supplementation in preventing preterm births in a population that also consumes food fortified by folates]

Czeizel, AE. “Periconceptional folic acid and multivitamin supplementation for the prevention of neural tube defects and other congenital abnormalities”. . vol. 85. 2009. pp. 260-268. [A good summary of the value of periconceptional folates in prevention of midline defects involving neural tube and neural crest cells]

Ionescu-Ittu, R, Marelli, AJ, Mackie, AS, Pilote, L. “Prevalence of severe congenital heart disease after folic acid fortification of grain products: time trend analysis in Quebec, Canada”. . vol. 338. 2009. pp. b1673[Confirmation of a population-wide benefit in reduction of midline congenital heart disease following food fortification with folates]