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Hematology Center
Megaloblastic Anemia

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MACROCYTOSIS

An increased MCV can be due to a number of reasons but careful review of the patient's history and blood smear can narrow the diagnostic possibilities. The differential can be divided into two broad categories based on RBC morphology.

Round macrocytosis-due to abnormal lipid composition of the erythrocyte membrane. Common etiologies include:

1. Alcoholism.
2. Liver Disease.
3. Renal Disease.
4. Hypothyroidism ("myxedema of the red cell").


Oval macrocytosis (macroovalocytes) is a sign of problem with cell DNA replication. The developing red cell has difficulty in undergoing cell division but RNA continues to be translated and transcribed into protein leading to growth of the cytoplasm while the nucleus lags behind. Often one or more cell division are skipped leading to a larger than normal cell. Common causes are:

1. Drug effect including cytotoxic chemotherapy (AZT now most common etiology of increased MCV).
2. Megaloblastic Anemias-Folate Deficiency or Vitamin B12 deficiency - Patients will have hypersegmented neutrophils on review of the peripheral smear.
3. Myelodysplasia - Patients often have hyposegmented neutrophils and abnormal platelet morphology.


Patients with RBC autoantibodies or cold agglutinins can have a spurious increase in the MCV due to red cell clumping in the automatic counters. Patients with increased reticulocyte counts can also have an increase MCV due to the large size of the reticulocyte (MCV = 160).

ABSORPTION AND METABOLISM OF VITAMIN B12 AND FOLATE

Folate-The body stores very little folate (four months) and maintenance of folate stores is dependent on adequate dietary intake. Folate is found in green leafy vegetables, fruits and liver. Folate is absorbed in the small bowel and circulates in a free form or loosely bond to albumin.

Vitamin B12- In contrast to folate the body stores copious amounts of vitamin B12 (2-6 years). This is fortunate as the absorption of vitamin B12 is complex and can be interrupted by a variety of mechanisms. Vitamin B12 is synthesized by microbes and the major dietary source is animal protein. When animal protein is ingested, vitamin B12 is freed from the protein and binds to "R proteins". The R protein-vitamin B12 complex travels to the duodenum where pancreatic enzymes destroy the R protein. This allows intrinsic factor (IF) to bind to vitamin B12. This IF-vitamin B12 complex is absorbed only in the last 1-2 feet of terminal ileum. Vitamin B12 binds to transcobalamin II and is delivered to tissues.

VITAMIN B12 AND FOLATE- METABOLIC PATHWAYS

Both vitamin B12 and folate are key components in the synthesis of DNA due to their role in conversion of uridine to thymidine. When methyltetrahydrofolate loses a methyl group to form tetrahyrodrofolate, vitamin B12 "shuttles" the methyl group to homocysteine converting it to methionine. Tetrahydrofolate is eventually converted to methylenetetrahyrofolate which is required for thymidine synthase. Vitamin B12 other role is a co-factor in the conversion of methymalonyl-CoA to succinyl-CoA.

CONSEQUENCES OF VITAMIN B12 OR FOLATE DEFICIENCY

When vitamin B12 or folate is deficient, thymidine synthase function is impaired and DNA synthesis is interrupted. As described above this leads to megaloblastic changes in all rapidly dividing cells. The inability to synthesized DNA leads to ineffectual erythropoiesis. There is often erythroid hyperplasia in the marrow but most of these immature cells die before reaching maturity. This process, intramedullary hemolysis, leads to the classic biochemical picture of hemolysis-raided LDH and indirect bilirubinemia. The LDH level is often in the 1,000's in patients with megaloblastic anemia. The lack of DNA synthesis affects the neutrophils leading to nuclear hypersegmentation. The anemia is of gradual onset and is often very well tolerated despite low hematocrits. Often a mild pancytopenia is seen but thrombocytopenia can be severe.

Other rapidly dividing tissue are influenced by the megaloblastic process. In the GI tract this can lead to atrophy of the luminal lining and further malabsorption.

As discussed further below, only vitamin B12 deficiency leads to neurological damage. The mechanism is unknown.

ETIOLOGIES OF FOLATE DEFICIENCY

Decreased intake

Increased requirements-Patients who are pregnant, have hemolytic anemia, or psoriasis have increased needs for folate which can cause them to rapidly develop folate deficiency if intake is not kept up.

Malabsorption

Drugs - Patient with underlying mild folate deficiency are more susceptible to trimethoprim/sulfa, pyrimethamine and methotrexate toxicity. Oral contraceptive and anticonvulsants lead to increase consumption of folate.

Alcohol- Alcohol affects several aspects of folate metabolism. Alcoholics have poor intake of folate. In addition, folate metabolism is interfered with leading to a functional folate deficiency. Alcoholics have an inability to mobilize folate stores and can have depleted tissue stores with normal serum levels of folate.

ETIOLOGIES OF VITAMIN B12 DEFICIENCY

Inadequate intake is rare but seen in very strict vegetarians (no eggs or milk).

Abnormal gastric events include being unable to dissociated vitamin B12 from food due to lack of stomach acid or enzymes. This is a recently recognized group of patients which may compose a very large subset of patients with vitamin B12 deficiency. 10-30% percent of patients with partial gastrectomy will develop vitamin B12 deficiency.

Deficient intrinsic factor most commonly occurs due to destruction of parietal cells by autoantibodies (pernicious anemia).

Abnormal small bowel events include pancreatic insufficiency, blind loops syndromes (bacterial absorbing vitamin B12-IF complexes) and patients infested with Diphyllobothrium latum.

Abnormal mucosal events including malabsorption syndromes and surgical removal of the terminal ileum.

APPROACH TO THE PATIENT WITH A MEGALOBLASTIC ANEMIA

1. Recognizing that a megaloblastic anemia is present.
2. Diagnosing vitamin B12 and or folate deficiency
3. Determining the underlying cause.
4. Therapy


DIAGNOSING VITAMIN B12 AND OR FOLATE DEFICIENCY

When a patient is believed to have a megaloblastic anemia or a process consistent with vitamin B12 deficiency, one should draw a serum vitamin B12 level, a red cell folate level (more sensitive indicative of tissue stores than serum folate) and since up to 30% of patients with megaloblastic anemia have concurrent iron deficiency, a serum ferritin.

One difficulty is that vitamin B12 levels are neither specific or sensitive for vitamin B12 deficiency. Low "normal" levels have been associated with anemia and neurological disease in up to 30% of patients. Recently there has been interest in measuring levels of serum homocysteine and methylmalonic acids. These precursors build up in vitamin B12 deficiency and appear to be more accurate indicator of tissue vitamin B12 deficiency. Increased levels of homocysteine and methylmalonic acid also are superior to vitamin B12 levels in predicting a response to vitamin B12 therapy.

DETERMINING THE UNDERLYING CAUSE

In the majority of patients with folate deficiency, one can determine the underlying cause by history. The key concern in vitamin B12 deficiency is determining a which point in the complex pathway of vitamin B12 absorption the "lesion" is. The Schilling test is a test of vitamin B12 absorption. Patients are given radiolabeled vitamin B12 orally and a large dose of vitamin B12 is given intravenously. The IV dose of vitamin B12 prevents binding any absorbed labeled vitamin B12 and this is excreted. The amount of excreted vitamin B12 is reflective of vitamin B12 absorption. The Schilling test is NOT a test of vitamin B12 deficiency but a tool to determine the etiology of the deficiency. The tradition Schilling test is call "stage I". If less than 8% of the labeled vitamin B12 is excreted then one can perform the Schilling test with a variety of diagnostic maneuvers to pinpoint the lesion. This includes giving intrinsic factor, pancreatic enzymes, or antibiotics.

The Schilling test has several shortcomings. One is it require patient cooperation in collecting the 24 hour urine sample. As noted above patients can have secondary malabsorption due to vitamin B12 deficiency. Several drugs including "slow K" will cause a false positive Schilling test. Finally the classic Schilling test will not detect abnormalities in patients with difficulties in disassociating vitamin B12 from food. The "food" Schilling test where labeled vitamin B12 is mixed with food has been proposed to detect this common group of patient.

Patients with pernicious anemia can be detected by assaying for autoantibodies but these tests can lack diagnostic specificity. Antibodies to IF are specific but not sensitive and antibodies to parietal cells are sensitive but not specific for pernicious anemia.

THERAPY

Patients with severe megaloblastic anemia need immediate therapy. One should quickly obtain serum vitamin B12 and red cell folate levels and then give 1-5 mg of folate and 1000 ug IM of vitamin B12. Patients should be treated daily with folate. Schedules for vitamin B12 replacement vary but a common approach to all is daily therapy for one week to rapidly build up stores and supply vitamin B12 to tissues, then weekly for a month, then monthly life-long. Patients with severe anemia should have increased reticulocyte by day three and increased hematocrit by day 5. Patients with alcoholism and folate deficiency can take up to three weeks to respond to folate therapy. It is routine to use IM injection to replace vitamin B12. Oral therapy with 1000 ug/day has been proposed. One should insure that their patient is replete before instituting oral therapy and monitor levels frequently since up to 30% of patients will not have adequate absorption to maintain their vitamin B12 stores.

Patients with low "normal" vitamin B12 levels should have serum homocysteine and methylmalonic acid concentrations assayed to determine the need for replacement.

Although patients will megaloblastic anemia often present with severe anemia, transfusion therapy is rarely indicated. Since the anemia is rapidly reversible with therapy there is little justification for exposing the patient to the risk of transfusion except if the patient is having life-threatening symptoms such as severe ischemia. A further hazard of transfusion is since some of these patients have high-output heart failure, overzealous transfusion may lead to pulmonary edema.

VITAMIN B12- NEUROLOGICAL CONSEQUENCES

Recently it has become clear the patients can have neurological damage due to vitamin B12 deficiency without anemia. In fact as many as 30% of patients with neurological disease due to vitamin B12 deficiency will have no or only subtle hematological symptoms. Patients with the most severe neurological manifestation often have mild hematological disease. Thus it is appearing that vitamin B12 deficiency may exhibit two different types of disease states in humans - hematological or neurological. Neurological symptoms are reversible if found early but those present for over a year slowly, if ever, improve.

The neurological symptoms include:

* Paresthesias-most often in fingers and toes. The most common symptom of vitamin B12 deficiency.
* Diminished vibratory sense
* Gait ataxia
* Increases deep tendon reflexes
* Memory loss
* Personality change
* Orthostatic hypotension


VITAMIN B12 AND THE ELDERLY

On routine screening as many as 10-23% of elderly patients will have low vitamin B12 levels. One study found that 14.5% had levels below 300 pg/ml with 56% of these patients having increased levels of homocysteine and methylmalonic acid indicative of tissue vitamin B12 deficiency. The most common mechanism is inability to absorb vitamin B12 from food. It is speculated the rapid rise in the use of H2 blockers will increase this problem in this patient population. Patients with dementia have lower levels of vitamin B12 then those without but treatment with vitamin B12 is often not effective, perhaps due to the long duration of the neurological damage. Studies are underway to examine the relationship of vitamin B12 deficiency to neurological disease in the elderly and the effects of early intervention.