	Patient Value	Reference Range
WBCs (× 10⁹/L)	3.2	4.5-11.0
RBCs (× 10¹²/L)	2.22	4.30-5.90
Hb (g/dL)	8.5	13.9-16.3
Hct (%)	27	39-55
MCV (fL)	121.6	80-100
MCH (pg)	38.3	25.4-34.6
MCHC (g/dL)	31.5	31-37
RDW (%)	18	11.5-13.5
Platelets (× 10⁹/L)	115	150-450
Reticulocytes (%)	1.8	0.5-1.5
WBC differential: unremarkable with the exception of hypersegmentation of neutrophils
RBC morphology: moderate anisocytosis, moderate poikilocytosis, macrocytes, oval macrocytes, few teardrop cells

Because of these findings, additional tests were ordered, with the following results:

• Serum vitamin B₁₂: decreased

• Serum folate: within reference range

• Serum methylmalonic acid: increased

1. Which of the CBC findings led the physician to order the vitamin assays?

2. Is the patient’s reticulocyte response adequate to compensate for the anemia?

3. Based on the available test results, what can you conclude about the cause of the patient’s anemia?

4. What additional testing would be helpful to diagnose the specific cause of this patient’s anemia?

Impaired deoxyribonucleic acid (DNA) metabolism causes systemic effects by impairing production of all rapidly dividing cells of the body. These are chiefly the cells of the skin, the epithelium of the gastrointestinal tract, and the hematopoietic tissues. Because these all must be replenished throughout life, any impairment of cell production is evident in these tissues first. Patients may experience symptoms in any of these systems, but the blood provides a ready tissue for analysis. The hematologic effects, especially megaloblastic anemia, have come to be recognized as the hallmark of the diseases affecting DNA metabolism.

Etiology

The root cause of megaloblastic anemia is impaired DNA synthesis. The anemia is named for the very large cells of the bone marrow that develop a distinctive morphology (see section on laboratory diagnosis) due to a reduction in the number of cell divisions. Megaloblastic anemia is one example of a macrocytic anemia. Box 20-1 shows the classification of macrocytic anemias. Understanding the etiology of megaloblastic anemia requires a review of DNA synthesis with particular attention to the roles of vitamin B₁₂ (cobalamin) and folic acid (folate).

BOX 20-1 Classification of Macrocytic Anemias by Cause

Impaired absorption (e.g., inflammatory bowel disease)

Impaired use due to drugs

Excessive loss with renal dialysis

Vitamin B₁₂ deficiency

Inadequate intake

Increased need

Impaired absorption

Failure to split from food

Failure to split from haptocorrin

Lack of intrinsic factor

Pernicious anemia

Helicobacter pylori infection

Gastrectomy

Hereditary intrinsic factor deficiency

General malabsorption (e.g., inflammatory bowel disease)

Inherited errors in absorption or transport

Imerslund-Gräsbeck syndrome

Transcobalamin deficiency

Competition for the vitamin

Diphyllobothrium latum (fish tapeworm) infection

Blind loop syndrome

Other causes of megaloblastosis

Myelodysplastic syndrome

Acute erythroleukemia (FAB-M6)

Congenital dyserythropoietic anemia

Reverse transcriptase inhibitors

Macrocytic, nonmegaloblastic anemias

Normal newborn status

Physiologic Roles of Vitamin B₁₂ and Folate

Vitamin B₁₂ (cobalamin) is an essential nutrient consisting of a tetrapyrrole (corrin) ring containing cobalt that is attached to 5,6-dimethylbenzimidazolyl ribonucleotide (Figure 20-1). Vitamin B₁₂ is a coenzyme in two biochemical reactions in humans. One is isomerization of methylmalonyl coenzyme A (CoA) to succinyl CoA, which requires vitamin B₁₂ (in the adenosylcobalamin form) as a cofactor and is catalyzed by the enzyme methylmalonyl CoA mutase (Figure 20-2). In the absence of vitamin B₁₂, the impaired activity of methylmalonyl CoA mutase leads to a high level of serum methylmalonic acid, which is useful for the diagnosis of vitamin B₁₂ deficiency (discussed in the section on laboratory diagnosis). The second reaction is the transfer of a methyl group from 5-methyltetrahydrofolate (5-methyl THF) to homocysteine, which thereby generates methionine. This reaction is catalyzed by the enzyme methionine synthase and uses vitamin B₁₂ (in the methylcobalamin form) as a coenzyme (discussed later in this section). Methylcobalamin is synthesized through reduction and methylation of vitamin B₁₂. This reaction represents the link between folate and vitamin B₁₂ coenzymes and appears to account for the requirement for both vitamins in normal erythropoiesis.^1,2

Figure 20-1 Structure of vitamin B₁₂ (cobalamin) and its analogs, hydroxycobalamin and cyanocobalamin (forms often found in food and supplements) and methylcobalamin and 5′-deoxyadenosylcobalamin (coenzyme forms). The basic structure of cobalamin includes a tetrapyrrole (corrin) ring with a central cobalt atom linked to 5,6-dimethylbenzimidazolyl ribonucleotide. (From Scott JM, Browne P: Megaloblastic anemia. In Caballero B, Allen L, Prentice A, editors: *Encyclopedia of human nutrition,* ed 2, Oxford, 2006, Academic Press, p 113.)

Figure 20-2 Role of vitamin B₁₂ in the metabolism of methylmalonyl coenzyme A (CoA). Vitamin B₁₂, in the 5′-deoxyadenosylcobalamin form, is a coenzyme in the isomerization of methylmalonyl CoA to succinyl CoA. The reaction is catalyzed by the enzyme methylmalonyl CoA mutase.

Folate is the general term used for any form of the vitamin folic acid. Folic acid is the synthetic form in supplements and fortified food. Folates consist of a pteridine ring attached to para-aminobenzoate with one or more glutamate residues (Figure 20-3). The function of folate is to transfer carbon units in the form of methyl groups from donors to receptors. In this capacity, folate plays an important role in the metabolism of amino acids and nucleotides. Deficiency of the vitamin leads to impaired cell replication and other metabolic alterations. Folate circulates in the blood predominantly as 5-methyl THF.³ 5-Methyl THF is metabolically inactive until it is demethylated to tetrahydrofolate (THF), whereupon folate-dependent reactions may take place.

Figure 20-3 Structure of synthetic folic acid and the naturally occurring forms of the vitamin. (From Scott JM, Browne P: Megaloblastic anemia. In Caballero B, Allen L, Prentice A, editors: *Encyclopedia of human nutrition,* ed 2, Oxford, 2006, Academic Press, p 114.)

Folate has an important role in DNA synthesis. As seen in Figure 20-4, within the cytoplasm of the cell, a methyl group is transferred from 5-methyl THF to homocysteine, which converts it to methionine and generates THF. This reaction is catalyzed by the enzyme methionine synthase and requires vitamin B₁₂ in the form of methylcobalamin as a cofactor. THF is then converted to 5,10-methylenetetrahydrofolate (5,10-methylene THF); the methyl group for this reaction comes from serine as it is converted to glycine. The methyl group of 5,10-methylene THF is then transferred to deoxyuridine monophosphate (dUMP), which converts it to deoxythymidine monophosphate (dTMP). This reaction is catalyzed by thymidylate synthetase and results in the conversion of 5,10-methylene THF to dihydrofolate (DHF). Deoxythymidine monophosphate is a precursor to deoxythymidine triphosphate (dTTP), which, like the other nucleotide triphosphates, is a building block of the DNA molecule. THF is regenerated by the conversion of DHF to THF by the enzyme DHF reductase. Because some of the folate is catabolized during the cycle, the regeneration of THF also requires additional 5-methyl THF from the plasma. It is important to note that once in the cell, folate is rapidly polyglutamated by the addition of one to six glutamic acid residues. This conjugation is required for retention of THF in the cell and it also promotes attachment of folate to enzymes.⁴

Figure 20-4 Role of folate and vitamin B₁₂ in DNA synthesis. Folate enters the cell as 5-methyltetrahydrofolate (5-methyl THF). In the cell, a methyl group is transferred from 5-methyl THF to homocysteine, converting it to methionine and generating tetrahydrofolate (THF). This reaction is catalyzed by methionine synthase and requires vitamin B₁₂ as a cofactor. THF is then converted to 5,10-methylene THF by the donation of a methyl group from serine. The methyl group of 5,10-methylene THF is then transferred to deoxyuridine monophosphate (dUMP), which converts it to deoxythymidine monophosphate (dTMP) and converts 5,10-methylene THF to dihydrofolate (DHF). This reaction is catalyzed by thymidylate synthetase. dTMP is a precursor of deoxythymidine triphosphate (dTTP), which is used to synthesize DNA. THF is regenerated by the conversion of DHF to THF by the enzyme DHF reductase. A deficiency of vitamin B₁₂ prevents the production of THF from 5-methyl THF; as a result, folate becomes metabolically trapped as 5-methyl THF. This constitutes the “folate trap.”

Defect in Megaloblastic Anemia due to Deficiency in Folate and Vitamin B₁₂

When either folate or vitamin B₁₂ is missing, thymidine nucleotide production for DNA synthesis is impaired. Folate deficiency has the more direct effect, ultimately preventing the methylation of dUMP. The effect of vitamin B₁₂ deficiency is more indirect, preventing the production of THF from 5-methyl THF. When vitamin B₁₂ is deficient, progressively more and more of the folate becomes metabolically trapped as 5-methyl THF. This constitutes what has been called the folate trap as 5-methyl THF accumulates and is unable to supply the folate cycle with THF. Some 5-methyl THF also leaks out of the cell if it is not readily polyglutamated. This results in a decrease in intracellular folate.⁵ In addition, when either folate or vitamin B₁₂ is deficient, homocysteine accumulates, because vitamin B₁₂ is unable to convert it to methionine (see Figure 20-4).

In this state of diminished thymidine availability, uridine is incorporated into DNA.6,7 The DNA repair process can remove the uridine, but without available thymidine, the repair process is unsuccessful. Although the DNA can unwind and replication can begin, at any point where a thymidine nucleotide is needed, there is essentially an empty space in the replicated DNA sequence, which results in many single-strand breaks. When excisions at opposing DNA strand sites coincide, double-strand breaks occur. Repeated DNA strand breaks lead to fragmentation of the DNA strand.⁴ The resulting DNA is nonfunctional, and the DNA replication process is incomplete. Cell division is halted, which results in either cell lysis or apoptosis⁸ of many erythroid cells. Cells that do not lyse are released into the circulation. The dependency of DNA production on folates has been used in cancer chemotherapy (Box 20-2).

BOX 20-2 Disruption of the Folate Cycle in Cancer Chemotherapy

The central role of folate in cell division makes it a target for chemotherapeutic drugs used to treat cancer. Folate analogues can be used to compete for folate in DNA production and result in impaired cell division. The cells in the cell cycle, such as cancer cells and other normally rapidly dividing cells such as epithelium and blood cells, are most susceptible to the drug interference. Methotrexate, used in the treatment of leukemia and arthritis, is an example of a folate antimetabolite drug. It has a higher affinity for dihydrofolate reductase than does tetrahydrofolate. Thus, methotrexate enters the folate cycle in preference to tetrahydrofolate, and the folate cycle is blocked by the drug. Methotrexate treatment typically is followed by what is known as leucovorin rescue. Leucovorin is a folic acid derivative that can be administered to counteract the effects of methotrexate or other folate antagonists.

In the case of red blood cell (RBC) development, these vitamin deficiencies result in ineffective hematopoiesis with increased apoptosis of erythroid progenitor cells. The remaining erythroid cells are larger than those cells that are normally seen during the final stages of erythropoiesis and their nuclei are immature-appearing compared with the cytoplasm (asynchrony). In contrast to the normally dense chromatin of comparable normoblasts, megaloblastic erythroid precursors have an open, finely stippled, reticular pattern.⁵ The nuclear changes seen in the megaloblastic cells are related to cell cycle delay, prolonged resting phase, and arrest in nuclear maturation. Electron microscopy has revealed that reduced synthesis of histones is also responsible for morphologic changes in the chromatin of megaloblastic cells, regardless of the causes.⁹ Because ribonucleic acid (RNA) contains uracil instead of thymidine residues, RNA function is not affected by vitamin B₁₂ or folate deficiency. Thus cytoplasmic development is not generally disturbed in these cells, which results in the asynchrony between cytoplasm and nucleus. Together, the accumulation of cells at earlier stages of differentiation and cells with increased size and immaturity result in the appearance of erythroid cells in the bone marrow that are pathognomonic of megaloblastic anemia.⁸ Due to the ineffective hematopoiesis, pancytopenia is also evident, with certain distinctive cellular changes (see section on laboratory diagnosis).

Other Causes of Megaloblastosis

Vitamin B₁₂ and folate deficiency are not the only causes of megaloblastic erythrocytes. Dysplastic erythroid cells in myelodysplastic syndrome (MDS) can also have megaloblastoid features (see Chapter 35). In MDS, the macrocytic erythrocytes and their progenitors characteristically show delayed cytoplasmic and nuclear maturation, including cytoplasmic vacuole formation, nuclear budding, multinucleation, and nuclear fragmentation, and thus may be distinguished from the megaloblastic RBCs seen in the vitamin deficiencies. In addition, nuclear-cytoplasmic asynchrony and megaloblastic RBCs may be seen in congenital dyserythropoietic anemia (CDA) types I and III (see Chapter 21). The CDAs are rare conditions that usually manifest in childhood and may be distinguished from the acquired causes of megaloblastosis by clinical history and morphologic differences. In CDA I internuclear chromatin bridging of erythroid cells or binucleated forms are observed, and in CDA III giant multinucleated erythroblasts are present. Another rare condition in which RBC precursors have a megaloblastic appearance is acute erythroleukemia, previously classified as FAB M6 (see Chapter 36). In this condition, the cells are macrocytic, and the immature appearance of the nuclear chromatin is similar to the more open appearance of the chromatin in megaloblasts. There are usually other aberrant findings in erythroleukemia, including an increase of myeloblasts in the bone marrow; however, an experienced morphologist can discern the subtle differences. Reverse transcriptase inhibitors, used to treat human immunodeficiency virus (HIV) infections, interfere with DNA production and may also lead to megaloblastic changes.¹⁰

Although the conditions described in this section are characterized by megaloblastic morphology, they are due to acquired or inherited mutations in progenitor cells or interference with DNA synthesis and are refractive to therapy with vitamin B₁₂ or folic acid.

Systemic Manifestations of Folate and Vitamin B₁₂ Deficiency

When DNA synthesis and subsequent cell division are impaired by lack of folate or vitamin B₁₂, megaloblastic anemia and its systemic manifestations develop. With either vitamin deficiency, patients may experience general symptoms related to the anemia (fatigue, weakness, and shortness of breath) and symptoms related to the alimentary tract. The loss of epithelium on the tongue results in a smooth surface and soreness (glossitis). Loss of epithelium along the gastrointestinal tract can result in gastritis, nausea, or constipation.

Although the blood pictures seen with the two vitamin deficiencies are indistinguishable, the clinical presentations vary. In vitamin B₁₂ deficiency, neurologic symptoms may be pronounced and may even occur in the absence of anemia.⁵ These include memory loss, numbness and tingling in toes and fingers, loss of balance, and further impairment of walking by loss of vibratory sense, especially in the lower limbs.¹¹ Neuropsychiatric symptoms may also be present, including personality changes and psychosis. These symptoms seem to be the result of demyelinization of the spinal cord and peripheral nerves, but the relationship of this demyelinization to vitamin B₁₂ deficiency is unclear. The role of increases in tumor necrosis factor-α, a neurotoxic agent, and decreases in epidermal growth factor, a neurotrophic agent, in the development of neurologic symptoms in vitamin B₁₂-deficient patients is being researched.^12,13

At one time, folate deficiency was believed to be more benign clinically than vitamin B₁₂ deficiency. Later research suggested that low levels of folate and the resulting high homocysteine levels were risk factors for cardiovascular disease.¹⁴ More recent research has not substantiated this association,^15–17 although some point out that high folate levels provide a cardioprotective effect in diabetic patients and certain ethnic populations.^18,19 The evidence at this time is unclear as to whether persistent suboptimal folate status may have a significant long-term health impact. In addition, there is evidence of depression, peripheral neuropathy, and psychosis related to folate deficiency.^20–22 Folate levels appear to influence the effectiveness of treatments for depression.²³ Folate deficiency during pregnancy can result in impaired formation of the fetal nervous system, resulting in neural tube defects such as spina bifida,²⁴ despite the fact that the fetus accumulates folate at the expense of the mother. Pregnancy requires a considerable increase in folate to fulfill the requirements related to rapid fetal growth, uterine expansion, placental maturation, and expanded blood volume.³ Ensuring adequate folate levels in women of childbearing age is particularly important because many women are likely to be unaware of their pregnancy during the first crucial weeks of fetal development. Fortification of the U.S. food supply with folic acid in grain and cereal products was mandated by the Food and Drug Administration in 1998 to lower the risk of neural tube defects in the unborn.

Causes of Vitamin Deficiencies

In general, vitamin deficiencies may arise because the vitamin is in relatively short supply, because use of the vitamin is impaired, or because excessive loss is occurring. Folate deficiency can be caused by all of these mechanisms.

Folate Deficiency

Inadequate Intake

The most common cause of folate deficiency is inadequate dietary intake.²⁵ Folate is ubiquitous in foods, but a generally poor diet can result in deficiency. Good sources of folate include leafy green vegetables, dried beans, liver, beef, fortified breakfast cereals, and some fruits, especially oranges.²⁶ Folates are heat labile, and overcooking of foods can diminish their nutritional value.³

Increased Need

Increased need for folate occurs during pregnancy and lactation when the mother must supply her own needs plus those of the fetus or infant. Infants and children also have increased need for folate during growth.³

Impaired Absorption

Folates are absorbed from the diet in the small intestine; however, only 50% of what is ingested is available for absorption.³ A rare autosomal recessive deficiency of a folate transporter protein (PCFT/HCP1) severely decreases intestinal absorption of folate.²⁷ Once across the intestinal cell, most folate is transported in the plasma as 5-methyl THF unbound to any specific carrier.¹¹ Its entry into cells, however, is carrier mediated.²⁸

Folate absorption may also be impaired by intestinal disease, especially sprue. Sprue is characterized by weakness, weight loss, and steatorrhea (fat in the feces), which is evidence that the intestine is not absorbing food properly. It is seen in the tropics (tropical sprue), where its cause is generally considered to be overgrowth of enteric pathogens.²⁹ Celiac disease (nontropical sprue) has been traced to intolerance of the gluten in some grains²⁹ (gluten-induced enteropathy) and can be controlled by eliminating wheat, barley, and rye products from the diet. Surgical resection of the small intestine and inflammatory bowel disease can also decrease folate absorption.

Impaired Use of Folate

Numerous drugs impair folate metabolism (Box 20-3).^30,31 Antiepileptic drugs are particularly known for this,³² and the result is macrocytosis with frank megaloblastic anemia. In most instances, folic acid supplementation is sufficient to override the impairment and allow the patient to continue therapy.³³

Excessive Loss of Folate

Physiologic loss of folate occurs through the kidney. The amount is small and not a cause of deficiency. Patients undergoing renal dialysis lose folate in the dialysate, however; thus supplemental folic acid is routinely provided to these individuals to prevent megaloblastic anemia.¹¹

Vitamin B₁₂ Deficiency

Inadequate Intake

True dietary deficiency of vitamin B₁₂ is possible for strict vegetarians (vegans) who do not eat meat, eggs, or dairy products. Although it is an essential vitamin for animals, plants cannot synthesize vitamin B₁₂, and it is not available from vegetable sources. The best dietary sources are animal products such as liver, milk, cheese, and eggs.²⁵

Increased Need

Increased need for vitamin B₁₂ occurs during pregnancy, lactation, and growth. Due to the vigorous cell replication, what would otherwise be a diet adequate in vitamin B₁₂ can become inadequate during these periods.

Impaired Absorption

Vitamin B₁₂ in food is released from food proteins primarily in the acid environment of the stomach, aided by pepsin, and is subsequently bound by a specific salivary protein, haptocorrin, also known as R-binder protein (Figure 20-5). In the small intestine, vitamin B₁₂ is released from haptocorrin by the action of pancreatic proteases, including trypsin. It is then bound by intrinsic factor, produced by the gastric parietal cells. Vitamin B₁₂ binding to intrinsic factor is required for absorption by the ileal cells (enterocytes) that possess receptors for the complex. These receptors are cubilin-amnionless complex, which binds the vitamin B₁₂–intrinsic factor compound, and megalin, a membrane transport protein.^34–37 Once in the enterocyte, the vitamin B₁₂ is then freed from intrinsic factor and bound to transcobalamin (previously called transcobalamin II) and released into the circulation. In the plasma, only 20% of the vitamin B₁₂ is bound to transcobalamin; the remaining 80% is bound to transcobalamin I and III, referred to as the haptocorrins.^34,38 The vitamin B₁₂–transcobalamin complex, termed holotranscobalamin, is the metabolically active form of vitamin B₁₂. Holotranscobalamin binds to specific receptors on the surfaces of many different types of cells and enters the cells by endocytosis, with subsequent release of vitamin B₁₂ from the carrier.³⁹ The body maintains a substantial reserve of absorbed vitamin B₁₂ in hepatocytes.

Figure 20-5 Normal absorption of vitamin B₁₂. Dietary vitamin B₁₂ (cobalamin, CBL) is protein (P) bound. In the stomach, pepsin and hydrochloric acid (HCl) secreted by parietal cells release CBL from P. CBL then binds salivary haptocorrin (R-binder protein, R) and remains bound until intestinal pancreatic proteases, including trypsin, catalyze its release. Parietal cells also secrete intrinsic factor (IF), which binds CBL in the duodenum. Cubilin-amnionless and megalin receptors in ileal enterocytes bind CBL-IF and release the CBL. Enterocytes produce transcobalamin (TC), which binds CBL and transports it through the portal circulation. Bone marrow pronormoblast membrane TC receptors (TC-R) bind CBL-TC and release the CBL, which is converted to methylcobalamin (methyl-CBL). Methyl-CBL is a coenzyme that supports homocysteine-methionine conversion. Hepatocyte TC-R receptors bind CBL-TC and release the CBL, which is moved to storage organelles or excreted through the biliary system.

The absorption of vitamin B₁₂ can be impaired by (1) failure to separate vitamin B₁₂ from food proteins in the stomach, (2) failure to separate vitamin B₁₂ from haptocorrin in the intestine, (3) lack of intrinsic factor, (4) malabsorption, and (5) competition for available vitamin.

Pernicious anemia

Pernicious anemia is an autoimmune disorder characterized by impaired absorption of vitamin B₁₂ due to a lack of intrinsic factor.⁴¹ This condition is called pernicious anemia because the disease was fatal before its cause was discovered. The incidence is approximately 25 per 100,000 persons older than 40 years of age.⁵ Pernicious anemia most often manifests in the sixth decade or later, but can also be found in children.

In pernicious anemia, autoimmune lymphocyte-mediated destruction of gastric parietal cells severely reduces the amount of intrinsic factor secreted in the stomach. Pathologic CD4 T cells inappropriately recognize and initiate an autoimmune response against the H⁺/K⁺–adenosine triphosphatase embedded in the membrane of the parietal cells.⁴² A chronic inflammatory infiltration follows, which extends into the wall of the stomach.⁴¹ Over a period of years and even decades, there is progressive development of atrophic gastritis resulting in the loss of the parietal cells with their secretory products, H⁺ and intrinsic factor. The loss of H⁺ production in the stomach constitutes achlorhydria. Low gastric acidity was previously an important diagnostic criterion for pernicious anemia. The absence of intrinsic factor can also be detected using the Schilling test. However, because the test requires a 24-hour urine collection and the use of radioactive cobalt in vitamin B₁₂ to trace absorption, it has fallen from favor, and safer diagnostic tests are preferred (see section on laboratory diagnosis).

Another feature of the autoimmune response in pernicious anemia is the production of antibodies to intrinsic factor ⁴³ and gastric parietal cells⁴⁴ that are detectable in serum. The most common antibody to intrinsic factor blocks the site on intrinsic factor where vitamin B₁₂ binds,⁴¹ which inhibits the formation of the intrinsic factor–vitamin B₁₂ complex and prevents the absorption of the vitamin. These blocking antibodies are present in about 70% of patients with pernicious anemia.⁴¹ Parietal cell antibodies are detectable in about 90% of patients with pernicious anemia.⁴¹

Other causes of lack of intrinsic factor

A lack of intrinsic factor may also be related to H. pylori infection. Left untreated, colonization of the gastric mucosa with H. pylori progresses until the parietal cells are entirely destroyed, a process involving both local and systemic immune processes.45,46 In addition, partial or total gastrectomy, which result in removal of intrinsic factor–producing parietal cells, invariably leads to vitamin B₁₂ deficiency.

Impaired absorption of vitamin B₁₂ can also be caused by hereditary intrinsic factor deficiency. This is a rare autosomal recessive disorder characterized by the absence or nonfunctionality of intrinsic factor. In contrast to the acquired forms of pernicious anemia, histology and gastric acidity are normal.³⁶

Malabsorption

General malabsorption of vitamin B₁₂ can be caused by the same conditions interfering with folate absorption, such as celiac disease, tropical sprue, and inflammatory bowel disease.

Inherited Errors of Vitamin B₁₂ Absorption and Transport

Imerslund-Gräsbeck syndrome is a rare autosomal recessive condition caused by mutations in the genes for either cubilin or amnionless. This defect results in decreased endocytosis of the intrinsic factor–vitamin B₁₂ complex by ileal cells. Transcobalamin deficiency is another rare autosomal recessive condition resulting in a deficiency of physiologically available vitamin B₁₂.36,47

Competition for Vitamin B₁₂

Competition for available vitamin B₁₂ in the intestine may come from intestinal organisms. The fish tapeworm Diphyllobothrium latum is able to split vitamin B₁₂ from intrinsic factor,⁴⁸ rendering the vitamin unavailable for host absorption. Also, blind loops, portions of the intestines that are stenotic as a result of surgery or inflammation, can become overgrown with intestinal bacteria that compete effectively with the host for available vitamin B₁₂.¹¹ In both of these cases, the host is unable to absorb sufficient vitamin B₁₂, and megaloblastic anemia results.

Laboratory Diagnosis

The tests used in the diagnosis of megaloblastic anemia include screening tests and specific diagnostic tests to identify the specific vitamin deficiency and perhaps its cause.

Screening Tests

Five tests used to screen for megaloblastic anemia are the complete blood count (CBC), white blood cell (WBC) manual differential, reticulocyte count, serum bilirubin, and lactate dehydrogenase.

Complete Blood Count

Slight macrocytosis often is the earliest sign of megaloblastic anemia. Patients with uncomplicated megaloblastic anemia are expected to have decreased hemoglobin and hematocrit values, pancytopenia, and reticulocytopenia. Megaloblastic anemia develops slowly, and the degree of anemia is often severe when first detected. Hemoglobin values of less than 7 or 8 g/dL are not unusual.⁴ When the hematocrit is less than 20%, erythroblasts with megaloblastic nuclei, including an occasional promegaloblast, may appear in the peripheral blood. The mean cell volume (MCV) is usually 100 to 150 fL and commonly is greater than 120 fL, although coexisting iron deficiency, thalassemia trait, or inflammation can prevent macrocytosis. The mean cell hemoglobin (MCH) is elevated by the increased volume of the cells, but the mean cell hemoglobin concentration (MCHC) is usually within the reference range, because hemoglobin production is unaffected. The RBC distribution width (RDW) is also elevated.

The characteristic morphologic findings of megaloblastic anemia in the peripheral blood include oval macrocytes (enlarged oval RBCs) (Figure 20-6) and hypersegmented neutrophils with six or more lobes (Figure 20-7).⁴⁹ Impaired cell production results in a low absolute reticulocyte count, especially in light of the severity of the anemia, and polychromasia is not seen on the peripheral blood film. Additional morphologic changes may include the presence of teardrop cells, RBC fragments, and microspherocytes. These smaller cells further increase the RDW. Nucleated RBCs, Howell-Jolly bodies, basophilic stippling, and Cabot rings may be observed.

Figure 20-6 Oval macrocytes, teardrop cells (dacryocytes), other red blood cell abnormalities, and a small lymphocyte for size comparison in megaloblastic anemia (peripheral blood, ×500).

Figure 20-7 Hypersegmented neutrophil in megaloblastic anemia (peripheral blood, ×1000).

White Blood Cell Manual Differential

Hypersegmentation of the neutrophils is essentially pathognomonic for megaloblastic anemia. It appears early in the course of the disease ⁵⁰ and may persist for up to 2 weeks after treatment is initiated.⁵ Hypersegmented neutrophils noted in the WBC differential report is a significant finding and requires a reporting rule that can be applied consistently, because even healthy individuals may have an occasional one. One such rule is to report hypersegmentation when there are at least 5 five-lobed neutrophils per 100 WBCs or at least 1 six-lobed neutrophil is noted.¹¹ Some laboratories perform a lobe count on 100 neutrophils and then calculate the mean. In megaloblastic anemia, the mean lobe count should be greater than 3.4.¹¹ The cause of the hypersegmentation is not understood, despite considerable investigation.⁵¹ More recent advances in the understanding of growth factors and their impact on transcription factors may yet solve this mystery. Nevertheless, a search for neutrophil hypersegmentation on a peripheral blood film constitutes an inexpensive yet sensitive screening test for megaloblastic anemia.

Bilirubin and Lactate Dehydrogenase Levels

Although generally considered a nutritional anemia, megaloblastic anemia is in one sense a hemolytic anemia, because the cells die during division in the marrow. This intramedullary cell death is known as ineffective erythropoiesis because many RBCs never enter the circulation—hence the decrease in reticulocytes in the peripheral blood. The usual signs of hemolysis are evident in the serum, including an elevation in the levels of total and indirect bilirubin and lactate dehydrogenase (predominantly RBC derived).

The constellation of findings including macrocytic anemia, moderate to marked pancytopenia, reticulocytopenia, oval macrocytes, hypersegmented neutrophils, plus increased levels of total and indirect bilirubin and lactate dehydrogenase justifies further testing to confirm a diagnosis of megaloblastic anemia and determine its cause. Occasionally, the classic findings may be obscured by coexisting conditions such as iron deficiency, which makes the diagnosis more challenging. Most hematologic aberrations do not appear until vitamin deficiency is fairly well advanced (Box 20-4).⁵²

BOX 20-4 Sequence of Development of Megaloblastic Anemias

1. Decrease in vitamin levels

2. Hypersegmentation of neutrophils in peripheral blood

3. Oval macrocytes in peripheral blood

4. Megaloblastosis in bone marrow

5. Anemia

Specific Diagnostic Tests

Bone Marrow Examination

Modern tests for vitamin deficiencies and autoimmune antibodies have made bone marrow examination an infrequently used diagnostic test for megaloblastic anemia. Nevertheless, it remains the reference confirmatory test to identify the megaloblastic appearance of the developing RBCs.

Megaloblastic, in contrast to macrocytic, anemia refers to specific morphologic changes in the developing RBCs. The cells are characterized by a nuclear-cytoplasmic asynchrony in which the cytoplasm matures as expected with increasing pinkness as hemoglobin accumulates. The nucleus lags behind, however, appearing younger than expected for the degree of maturity of the cytoplasm (Figure 20-8). This asynchrony is most striking at the stage of the polychromatophilic normoblast. The cytoplasm appears pinkish blue as expected for that stage, but the nuclear chromatin remains more open than expected, similar to that in the nucleus of a basophilic normoblast. Overall, the marrow is hypercellular, with a myeloid-to-erythroid ratio of about 1 : 1 by virtue of the increased erythropoietic activity. The hematopoiesis is ineffective, however, and although cell production in the bone marrow is increased, the death of cells in the marrow results in peripheral pancytopenia.

Figure 20-8 Erythroid precursors in megaloblastic anemia (bone marrow, ×1000).

The WBCs are also affected in megaloblastic anemia and appear larger than normal. This is most evident in metamyelocytes and bands, because in the usual development of neutrophils, the cells should be getting smaller at these stages. The effect creates what are called “giant” metamyelocytes (Figure 20-9) and bands.

Figure 20-9 Giant metamyelocyte in megaloblastic anemia (bone marrow, original magnification ×1000).

Megakaryocytes do not show consistent changes in megaloblastic anemia. They may be either increased or decreased in number and may show diminished lobulation. The latter finding is not consistently seen, however, and even when present is difficult to assess.

Assays for Folate, Vitamin B₁₂, Methylmalonic Acid, and Homocysteine

Although bone marrow aspiration is confirmatory for megaloblastosis, the invasiveness of the procedure and its expense mean that other testing is performed more often than a bone marrow examination. Furthermore, the confirmation of megaloblastic morphology in the marrow does not identify its cause. Tests for serum levels of folate and vitamin B₁₂ are readily available using immunoassay. RBC folate levels may also be measured. Unlike serum folate levels, which fluctuate with diet, RBC folate values are stable and may be a more accurate reflection of true folate status ⁵³; however, current RBC folate tests have less than optimal sensitivity and specificity and have not been validated in actual patients with normal and deficient folate levels. Thus the serum folate level is preferred over RBC folate level in the United States as the initial test for evaluation of folate deficiency.⁵

Some laboratories conduct a reflexive assay for methylmalonic acid if vitamin B₁₂ levels are low. As indicated previously, in addition to playing a role in folate metabolism, vitamin B₁₂ is a cofactor in the conversion of methylmalonyl CoA to succinyl CoA by the enzyme methylmalonyl CoA mutase (see Figure 20-2). If vitamin B₁₂ is deficient, methylmalonyl CoA accumulates. Some of it hydrolyzes to methylmalonic acid, and the increase can be detected in serum and urine. Because methylmalonic acid is also elevated in patients with impaired renal function, increased levels cannot be definitively related to vitamin B₁₂ deficiency.²⁵ Methylmalonic acid is assayed by gas chromatography–tandem mass spectrometry.

Homocysteine levels are affected by folate and vitamin B₁₂. 5-Methyl THF donates a methyl group to homocysteine in the generation of methionine. This process uses vitamin B₁₂ as a coenzyme (see Figure 20-4). Thus, a deficiency in either folate or vitamin B₁₂ results in elevated levels of homocysteine. Total homocysteine can be measured in either plasma or serum. Homocysteine may be assayed by gas chromatography–mass spectrometry, high-performance liquid chromatography, or fluorescence polarization immunoassay. Homocysteine levels are also elevated in patients with renal failure and dehydration.

Gastric Analysis

Gastric analysis may be used to confirm achlorhydria, an expected finding in pernicious anemia. Achlorhydria occurs in other conditions, however, including natural aging. When other causes of vitamin B₁₂ deficiency have been eliminated, a finding of achlorhydria is supportive, although not diagnostic, of pernicious anemia. The H⁺ concentration is determined by pH measurement.

Antibody Assays

Antibodies to intrinsic factor and parietal cells can be detected in the serum of most patients with pernicious anemia. Various immunoassays can detect intrinsic factor–blocking antibodies; parietal cell antibodies can be detected by indirect fluorescent antibody techniques or enzyme-linked immunosorbent assays.

Holotranscobalamin Assay

Holotranscobalamin is the metabolically active form of vitamin B₁₂. Until recently, methods for measuring holotranscobalamin were manual and not suitable for use in clinical laboratories. Newer, more rapid immunoassays using monoclonal antibodies specific for holotranscobalamin have been developed in the past several years that are both sensitive and specific.54,55

Deoxyuridine Suppression Test

The principle of the deoxyuridine suppression test is that the preincubation of normal bone marrow with deoxyuridine will suppress the subsequent incorporation of labeled thymidine into DNA, because the normal cells can successfully methylate the uridine into thymidine. However, in patients with either a vitamin B₁₂ or a folate deficiency, this suppression is abnormally low. By adding either vitamin B₁₂ or folate to the test cells, one can determine whether the inadequate suppression is caused by vitamin B₁₂ or folate deficiency.56–58 Although micromethods have been developed, the necessity of using bone marrow tissue and the complexity of the test make it impractical for clinical testing.

Stool Analysis for Parasites

When vitamin B₁₂ is found to be deficient, a stool analysis for eggs or proglottids of the fish tapeworm D. latum may be part of the diagnostic workup.

See Table 20-1 for a summary of laboratory tests used to diagnose vitamin B₁₂ and folate deficiency.

TABLE 20-1

Laboratory Tests Used to Diagnose Vitamin B₁₂ and Folate Deficiency

		Folate Deficiency	Vitamin B₁₂ Deficiency
Screening tests	Complete blood count	↓ Hb, Hct, RBCs, WBCs, PLTs ↑ MCV, MCH	Same as Folate Deficiency
	Manual differential count	Hypersegmented neutrophils, oval macrocytes, anisocytosis, poikilocytosis, RBC inclusions	Same as Folate Deficiency
	Absolute reticulocyte count	↓	↓
	Serum total and indirect bilirubin	↑	↑
	Serum lactate dehydrogenase	↑	↑
Specific diagnostic tests	Bone marrow examination*	Erythroid hyperplasia (ineffective) Presence of megaloblasts	Same as Folate Deficiency
	Serum vitamin B₁₂	N	↓
	Serum folate	↓	N or ↑^†
	RBC folate	↓	N or ↓^†
	Serum methylmalonic acid	N	↑
	Serum/plasma homocysteine	↑	↑
	Antibodies to intrinsic factor and gastric parietal cells	Absent	Present in pernicious anemia
	Gastric analysis*	N	Achlorhydria in pernicious anemia
	Holotranscobalamin assay^‡	N	↓
	Stool analysis for parasites	Negative	Diphyllobothrium latum may be the cause of deficiency