Chapter 18 -

Anemias Caused by Defects of DNA Metabolism

Impaired deoxyribonucleic acid (DNA) metabolism causes systemic effects by impairing the 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 (discussed in Laboratory Diagnosis) because of a reduction in the number of cell divisions. Megaloblastic anemia is one example of a macrocytic anemia. Understanding the cause of megaloblastic anemia requires a review of DNA synthesis with particular attention to the roles of vitamin B12 (cobalamin) and folic acid (folate).

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. It has various analogs, including hydroxycobalamin and cyanocobalamin (forms often found in food and supplements) and coenzyme forms, methylcobalamin and 5'-deoxyadenosylcobalamin. 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 deoxyadenosylcobalamin form) as a cofactor and is catalyzed by the enzyme methylmalonyl CoA mutase. In the absence of vitamin B12, the impaired activity of methylmalonyl CoA mutase leads to a high level of serum methylmalonic acid (MMA), which is useful for the diagnosis of vitamin B12 deficiency. 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. 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.

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. 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. 5-Methyl THF is metabolically inactive until it is demethylated to tetrahydrofolate (THF), whereupon folate-dependent reactions may take place.

Folate has an important role in DNA synthesis. 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 synthase 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 dihydrofolate reductase. Because some of the folate is catabolized during the cycle, the regeneration of THF also requires additional 5-methyl THF from the plasma. 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.

Defect in Megaloblastic Anemia Caused by Deficiency in Folate and Vitamin B12

When either folate or vitamin B12 is deficient, 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. In addition, when either folate or vitamin B12 is deficient, homocysteine accumulates because methionine synthase is unable to convert it to methionine without vitamin B12 as a cofactor.

In this state of diminished thymidine availability, uridine is incorporated into DNA. The DNA repair process can remove the uridine, but without available thymidine to replace it, 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. The resulting DNA is nonfunctional, and the DNA replication process is incomplete. Cell division is halted, resulting in either cell lysis or apoptosis of many erythroid progenitors and precursors within the bone marrow. Cells that survive continue the abnormal maturation with a fewer number of red blood cells (RBCs) released into the circulation. This abnormal blood cell development is called ineffective hematopoiesis. The dependency of DNA production on folate has been used in cancer chemotherapy.

In addition to the increased apoptosis of erythroid progenitor and precursor cells in the bone marrow discussed earlier, the remaining erythroid precursors are larger than normally seen during the final stages of erythropoiesis, and their nuclei are immature-appearing compared with the cytoplasm. In contrast to the normally dense chromatin of comparable normoblasts, the nuclei of megaloblastic erythroid precursors have an open, finely stippled, reticular pattern. The nuclear changes seen in the megaloblastic erythroid precursors are related to cell cycle delay, prolonged resting phase, and arrest in nuclear maturation. Electron microscopy reveals that reduced synthesis of histones is also responsible for morphologic changes in the chromatin of megaloblastic erythroid precursors. Ribonucleic acid (RNA) function is not affected by vitamin B12 or folate deficiency because RNA contains uracil instead of thymidine nucleotides, so cytoplasmic development progresses normally. The slower maturation rate of the nucleus compared with the cytoplasm is called nuclear-cytoplasmic asynchrony. Together the accumulation of cells with nuclei at earlier stages of development and cells with increased diameter and immaturity result in the appearance of erythroid precursors in the bone marrow that are pathognomonic of megaloblastic anemia. Because ineffective hematopoiesis affects all three blood cell lineages, pancytopenia is also evident, with certain distinctive cellular changes.

Other Causes of Megaloblastosis

Vitamin B12 and folate deficiency are not the only causes of megaloblastic erythroid precursors. Dysplastic erythroid precursors in myelodysplastic syndrome (MDS) can also have megaloblastoid features. In MDS, however, the macrocytic erythrocytes and their precursors 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 erythroid precursors seen in the vitamin deficiencies. In addition, nuclear-cytoplasmic asynchrony and megaloblastic erythroid precursors may be seen in congenital dyserythropoietic anemia (CDA) types I and III. 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 precursors or binucleated forms are observed, and in CDA III, giant multinucleated erythroblasts are present.

Another rare condition in which erythroid precursors have a megaloblastic appearance is acute erythroid leukemia, previously classified as FAB M6. In this condition, the erythroblasts 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 erythroid leukemia, 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.

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 anemia (fatigue, weakness, and shortness of breath) and symptoms related to the alimentary tract. 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. After dietary deficiency or malabsorption begins, it takes a few years to develop a vitamin B12 deficiency but only a few months to develop a folate deficiency, reflecting the storage capacity of each vitamin in the body. In vitamin B12 deficiency, neurologic symptoms may be pronounced and may even occur in the absence of anemia. 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. Neuropsychiatric symptoms may also be present, including personality changes and psychosis. These symptoms seem to be the result of demyelination of the spinal cord and peripheral nerves, but the relationship of this demyelination to vitamin B12 deficiency is unclear. The roles of increases in tumor necrosis factor-alpha, a neurotoxic agent, and decreases in epidermal growth factor, a neurotrophic agent, in the development of neurologic symptoms in vitamin B12-deficient patients are being researched.

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. More recent research has provided mixed results, with studies both refuting this association and substantiating the association between high circulating homocysteine levels and the risk of cardiovascular disease. Several studies suggest that high folate levels provide a cardioprotective effect in diabetic patients and certain ethnic populations. 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. Folate levels appear to influence the effectiveness of treatments for depression.

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 of excessive loss. Folate deficiency can be caused by all of these mechanisms.

Folate Deficiency

Inadequate Intake

Folate is synthesized by microorganisms and higher plants. 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. Folates are heat-labile, and overcooking of foods can diminish their nutritional value.

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.

Impaired Absorption

Food folates must be hydrolyzed in the gut before absorption in the small intestine; however, only 50% of what is ingested is available for absorption. A rare autosomal recessive deficiency of a folate transporter protein (PCFT) severely decreases intestinal absorption of folate. Once across the intestinal cell, most folate is transported in the plasma as 5-methyl THF unbound to any specific carrier. Its entry into cells, however, is by both carrier systems and receptors.

Folate absorption may also be impaired by intestinal disease, especially sprue and celiac disease. 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. Celiac disease (nontropical sprue) has been traced to intolerance of gluten in some grains (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 decrease absorption of folic acid or impair folate metabolism. Antineoplastic, antibacterial, and antiseizure agents are particularly known for this, and the result is macrocytosis with frank megaloblastic anemia. Because folate deficiency results in inhibition of cell replication, several anticancer drugs, including methotrexate, are folate inhibitors. In most instances, supplementation with folic acid or reduced folic acid (in the form of folinic acid) is sufficient to override the impairment and allow the patient to continue therapy.

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.

Vitamin B12 Deficiency

Inadequate Intake

Although true dietary deficiency of vitamin B12 is rare, this condition 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 thus it is not available from vegetable sources. The best dietary sources are animal products such as liver, dairy products, fish, shellfish, and eggs. In contrast to the heat-labile folate, vitamin B12 is not destroyed by cooking.

Increased Need

Increased need for vitamin B12 occurs during pregnancy, lactation, and growth. Because of 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 protein or transcobalamin I. 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, which is produced by gastric parietal cells. Vitamin B12 binding to intrinsic factor is required for absorption by ileal enterocytes that possess receptors for the complex. These receptors are cubilin-amnionless complex, collectively known as cubam, which binds the vitamin B12-intrinsic factor complex, and megalin, a membrane transport protein. 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 10% to 30% of the vitamin B12 is bound to transcobalamin; the remaining 75% is bound to transcobalamin I and III, referred to as the haptocorrins. The vitamin B12-transcobalamin complex, termed holotranscobalamin (holoTC), is the metabolically active form of vitamin B12. Holotranscobalamin binds to specific receptors on the surfaces of many different cells and enters the cells by endocytosis, with subsequent release of vitamin B12 from the carrier. The body maintains a substantial reserve of absorbed vitamin B12 in hepatocytes and kidney cells.

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.

5. Competition for available vitamin B12.

Failure to Separate Vitamin B12 from Food Proteins

A condition known as food-cobalamin malabsorption is characterized by hypochlorhydria and the resulting inability of the body to release vitamin B12 from food or intestinal transport proteins for subsequent binding to intrinsic factor. Food-cobalamin malabsorption is caused primarily by the reduced gastric acidity in atrophic gastritis or atrophy of the stomach lining that often occurs with increasing age. It also occurs with gastric bypass surgery and the long-term use of histamine type 2 receptor blockers and proton pump inhibitors, which reduce gastric acidity for the treatment of ulcers and gastroesophageal reflux disease.

Failure to Separate Vitamin B12 from Haptocorrin

Lack of gastric acidity or lack of trypsin as a result of chronic pancreatic disease can prevent vitamin B12 absorption because the vitamin remains bound to haptocorrin in the intestine and unavailable to intrinsic factor.

Lack of Intrinsic Factor

Lack of intrinsic factor constitutes a significant cause of impaired vitamin B12 absorption. It is most commonly a result of autoimmune disease, as in pernicious anemia, but can also result from hereditary intrinsic factor deficiency or loss of parietal cells in Helicobacter pylori infection or after total or partial gastrectomy.

Pernicious Anemia

Pernicious anemia is an autoimmune disorder characterized by impaired absorption of vitamin B12 because of an intrinsic factor deficiency. This condition is called pernicious anemia because the disease was fatal before its cause was discovered. The incidence per year is roughly 25 new cases per 100,000 persons older than 40 years of age. Pernicious anemia most often manifests in the sixth decade or later but can also be found in children. Patients with pernicious anemia have an increased risk of developing gastric tumors.

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+ ATPase embedded in the membrane of parietal cells. A chronic inflammatory infiltration follows, which extends into the wall of the stomach. 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. Serum gastrin levels can be markedly elevated as a result of the gastric achlorhydria. 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, safer diagnostic tests are currently used.

Another feature of the autoimmune response in pernicious anemia is the production of antibodies to intrinsic factor and gastric parietal cells that are detectable in serum. The most common antibody to intrinsic factor blocks the site on intrinsic factor where vitamin B12 binds, which inhibits the formation of the intrinsic factor–vitamin B12 complex and prevents the absorption of the vitamin. These blocking antibodies are present in serum or gastric fluid in 70% to 90% of patients with pernicious anemia, and their presence is highly specific for the disease. Parietal cell antibodies are detectable in the serum of 50% to 90% of patients with pernicious anemia.

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 mechanisms. In addition, partial or total gastrectomy, which results 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 impaired function of intrinsic factor. In contrast to the acquired forms of pernicious anemia, histology and gastric acidity are normal.

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 enterocytes. Transcobalamin deficiency is another rare autosomal recessive condition resulting in a deficiency of physiologically available vitamin B12.

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, 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. In both 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), reticulocyte count, white blood cell (WBC) manual differential, serum bilirubin, and lactate dehydrogenase.

Complete Blood Count and Reticulocyte 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. 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 interval because hemoglobin production is unaffected. The red blood cell distribution width (RDW) is also elevated.

The characteristic morphologic findings of megaloblastic anemia in the peripheral blood include oval macrocytes (enlarged oval RBCs) and hypersegmented neutrophils with six or more lobes. Impaired cell production results in a low absolute reticulocyte count, especially in light of the severity of the anemia, and polychromasia is not observed on the peripheral blood film. Additional morphologic changes may include the presence of teardrop cells (dacryocytes), RBC fragments, and microspherocytes. These smaller cells further increase the RDW. The presence of schistocytes sometimes leads to a paradoxically lower MCV than is seen in less severe cases. These erythrocyte changes reflect the severity of the dyserythropoiesis and should not be taken as evidence of microangiopathic hemolysis. Nucleated RBCs, Howell-Jolly bodies, basophilic stippling, and Cabot rings may also be observed.

White Blood Cell Manual Differential Count

Hypersegmentation of neutrophils is essentially pathognomonic for megaloblastic anemia. It appears early in the course of the disease and may persist for up to 2 weeks after treatment is initiated. Hypersegmented neutrophils noted in the WBC differential report are a significant finding and require 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. 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. The cause of the hypersegmentation is not understood, despite considerable investigation. 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 many erythroid progenitors and precursors die during division in the bone marrow, many RBCs never enter the circulation (ineffective hematopoiesis), so a decrease in reticulocytes occurs in the peripheral blood. The usual signs of hemolysis are evident in the serum, including an elevation in 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, and 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 or thalassemia, which makes the diagnosis more challenging. Most hematologic aberrations do not appear until vitamin deficiency is fairly well advanced.

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 erythroid precursors.

Megaloblastic, in contrast to macrocytic, anemia refers to specific morphologic changes in the developing erythroid precursors. The cells are characterized by 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. This asynchrony is most striking at the stage of the polychromatic 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 bone marrow is hypercellular, with a reduced myeloid-to-erythroid ratio of about 1:1 by virtue of the increased erythropoietic activity. Hematopoiesis is ineffective, however, and although cell production in the bone marrow is increased, the apoptosis of hematopoietic cells in the marrow results in peripheral pancytopenia.

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 "giant" metamyelocytes and bands.

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, it 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; serum vitamin B12 may also be assayed by competitive binding chemiluminescence. However, there are a number of interferences with these assays that can cause false increased and decreased results; reflexive testing to MMA and homocysteine can increase diagnostic accuracy. 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; 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.

Some laboratories conduct a reflexive assay for MMA 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. 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 MMA is also elevated in patients with impaired renal function, the test is not specific, and thus increased levels cannot be definitively related to vitamin B12 deficiency. Methylmalonic acid is assayed by gas chromatography-tandem mass spectrometry.

Homocysteine levels are affected by deficiencies in either folate or vitamin B12. 5-Methyl THF donates a methyl group to homocysteine in the generation of methionine. This reaction uses vitamin B12 as a coenzyme. 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 and Serum Gastrin

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.

As a result of the gastric achlorhydria, serum gastrin levels can be markedly elevated. Serum gastrin is measured by immunoassay, including chemiluminescent immunometric assays.

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. Anti-intrinsic factor antibodies are highly specific and confirmatory for pernicious anemia, but their absence does not rule out the condition. The test for parietal cell antibodies is non-specific and not clinically useful for the diagnosis of pernicious anemia.

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. Recent studies support the use of holotranscobalamin to detect vitamin B12 deficiency and recommend its use in screening for metabolic vitamin B12 deficiency.

Stool Analysis for Parasites

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

MACROCYTIC NONMEGALOBLASTIC ANEMIAS

The macrocytic nonmegaloblastic anemias are macrocytic anemias in which DNA synthesis is unimpaired. The macrocytosis tends to be mild; the MCV usually ranges from 100 to 110 fL and rarely exceeds 120 fL. Patients with nonmegaloblastic, macrocytic anemia lack hypersegmented neutrophils and oval macrocytes in the peripheral blood and megaloblasts in the bone marrow. Macrocytosis may be physiologically normal, as in the newborn, or the result of a pathologic condition, as in liver disease, chronic alcoholism, or bone marrow failure. Reticulocytosis is a common cause of macrocytosis.

TREATMENT

Treatment should be directed at the specific vitamin deficiency established by the diagnostic tests and should include addressing the cause of the deficiency (e.g., better nutrition, treatment for D. latum) if possible. Vitamin B12 may be administered intramuscularly to treat pernicious anemia to bypass the need for intrinsic factor. High-dose oral vitamin B12 treatment is increasingly popular in the treatment of pernicious anemia. Regardless of the treatment modality, those with pernicious anemia or malabsorption must have lifelong vitamin replacement therapy.

Folic acid can be administered orally. The inappropriate treatment of vitamin B12 deficiency with folic acid improves the anemia but does not correct or stop the progress of the neurologic damage, which may advance to an irreversible state. Thus, proper diagnosis before treatment is important. Iron is often supplemented concurrently to support the rapid cell production that accompanies effective treatment. When proper treatment is initiated, the body’s response is prompt and brisk and can be used to confirm the accuracy of the diagnosis. Bone marrow morphology will begin to revert to a normoblastic appearance within a few hours of treatment. A substantial reticulocyte response is apparent at about 1 week, with hemoglobin increasing toward normal levels in about 3 weeks.11 Hypersegmented neutrophils disappear from the peripheral blood within 2 weeks of initiation of treatment. Thus with proper treatment, hematologic parameters may return to normal within 3 to 6 weeks and correction of the megaloblastic anemia may occur in 6 to 8 weeks.