REVIEW CME MOC Daniel S. Socha, MD Sherwin I. DeSouza, MD Aron Flagg, MD Department of Laboratory Medicine, Department of and Medical Oncology, Department of Pediatric Hematology and Oncology, Robert J. Tomsich Pathology and Laboratory Taussig Cancer Institute, Cleveland Clinic Yale School of Medicine, New Haven, CT Medicine Institute, Cleveland Clinic Mikkael Sekeres, MD, MS Heesun J. Rogers, MD, PhD Department of Hematology and Medical Oncology and Department Department of Laboratory Medicine, Robert J. Tomsich Pathology and of Translational Hematology and Oncology Research, Taussig Laboratory Medicine Institute,Cleveland Clinic; Assistant Professor, Cancer Institute, Cleveland Clinic; Professor, Cleveland Clinic Lerner Cleveland Clinic Lerner College of Medicine of Case Western Reserve College of Medicine of Case Western Reserve, Cleveland, OH University, Cleveland, OH Severe megaloblastic : defi ciency and other causes

ABSTRACT ot all megaloblastic result N from vitamin defi ciency, but most do. De- Megaloblastic anemia causes from termining the underlying cause and initiating ineffective production and intramedullary prompt treatment are critical, as prognosis and . The most common causes are (vitamin management differ among the various condi- B9) defi ciency and cobalamin () defi ciency. tions. Megaloblastic anemia can be diagnosed based on char- This article describes the pathobiology, acteristic morphologic and laboratory fi ndings. However, presentation, evaluation, and treatment of se- other benign and neoplastic diseases need to be con- vere megaloblastic anemia and its 2 most com- sidered, particularly in severe cases. Therapy involves mon causes: folate (vitamin B9) and cobalamin treating the underlying cause—eg, with vitamin supple- (vitamin B12) defi ciency, with 2 representative mentation in cases of defi ciency, or with discontinuation case studies. of a suspected medication. ■ MEGALOBLASTIC ANEMIA OVERVIEW KEY POINTS Megaloblastic anemia is caused by defec- The hallmark of megaloblastic anemia is macrocytic ane- tive DNA synthesis involving hematopoi- mia ( > 100 fL), often associ- etic precursors, resulting in ineffective red blood cell production (erythropoiesis) and ated with other cytopenias. intramedullary hemolysis. Macrocytic ane- mia with increased mean corpuscular vol- Dysplastic features may be present and can be diffi cult to ume (MCV), defi ned as more than 100 fL, differentiate from myelodysplastic syndrome. is the hallmark of megaloblastic anemia, but leukopenia and thrombocytopenia are also Megaloblastic anemia is most commonly caused by folate frequently present. defi ciency from dietary defi ciency, , or malab- The incidence of is as high as 4% in the general population, but megalo- sorption syndromes or by vitamin B12 defi ciency, usually due to pernicious anemia. blastic anemia accounts for only a small frac- tion.1 Nonmegaloblastic causes of macrocytic anemia include ethanol abuse, myelodysplas- Both vitamin defi ciencies cause hematologic signs and tic syndrome, , hypothyroid- 2,3 symptoms of anemia; vitamin B12 defi ciency also causes ism, liver disease, and drugs. Although these neurologic symptoms. causes are associated with increased MCV, they do not lead to the other features of mega- Oral supplementation is available for both vitamin loblastic anemia. The most frequent causes of megaloblastic defi ciencies; intramuscular vitamin B12 supplementa- tion should be used in cases involving severe neurologic anemia are defi ciencies of vitamin 9B (folate) or vitamin B (cobalamin) (Table 1). Less- symptoms or gastric or bowel resection. 12 frequent causes include congenital disorders doi:10.3949/ccjm.87a.19072 (inborn errors of metabolism), drugs (particu-

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TABLE 1

Characteristics of vitamin B12 vs folate defi ciency

Vitamin B12 defi ciency Folate defi ciency Etiology Lack of : pernicious anemia Dietary defi ciency: alcoholism, countries without food fortifi cation : celiac disease, prior gastric or ileal surgery Malabsorption: developed countries Dietary defi ciency less common Increased demand: , , eczema Clinical Hematologic fi ndings: cytopenias Hematologic fi ndings: cytopenias presentation Neuropsychiatric symptoms: paresthesias, decreased proprioception and vibratory sense, dementia, confusion Evaluation Clinical history and physical examination: symptoms sec- Clinical history and physical examination:

ondary to anemia and hemolysis, neurologic symptoms similar to vitamin B12 defi ciency, except no neurologic symptoms Laboratory testing: serum vitamin B12, methylmalonic acid, homocysteine, antiparietal cell and anti-intrinsic factor Laboratory testing: serum folate, red blood antibodies, serum gastrin cell folate, methylmalonic acid, homocysteine Gastric biopsy for suspected pernicious anemia Differential Other macrocytic anemias without megaloblastic features: Other macrocytic anemias without megalo- diagnosis liver disease, thyroid dysfunction, alcohol abuse blastic features: liver disease, thyroid dysfunc- tion, alcohol abuse Myelodysplastic syndrome, acute myeloid leukemia Myelodysplastic syndrome, acute myeloid Nitrous oxide exposure leukemia Medication effect Medication effect

Treatment Parenteral vitamin B12 1–2 times per week until symptoms Oral folate daily improve, then monthly

High-dose oral vitamin B12 daily Monitoring Clinical follow-up for improvement of neurologic symptoms Monitor hematologic response: and follow-up complete blood cell count Monitor hematologic response: complete blood cell count Pernicious anemia: consider monitoring methylmalonic acid

larly chemotherapeutics and folate antago- Folate defi ciency has 3 main causes4,5: nists), micronutrient defi ciencies, and nitrous • Reduced intake from diets lacking folate oxide exposure.4,5 (rare in countries with vitamin fortifi cation) and alcoholism (see Case 1) ■ • Decreased absorption from disorders affect- Folate is found in green leafy vegetables, fruits, ing nutrient absorption in the small bowel, nuts, eggs, and meats. Normal body stores of eg, celiac disease, infl ammatory bowel dis- folate are 5 to 30 mg. The recommended daily ease, and tropical sprue allowance depends on age, sex, and pregnancy • Increased demand from pregnancy, hemo- status, but is generally 400 μg in adults and lytic anemia, puberty, and eczematous con- 600 μg during pregnancy.6 ditions.

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■ TABLE 2 Vitamin B is produced by microorganisms 12 Causes of vitamin B defi ciency and is found almost exclusively in foods of 12 animal origin. Normal body stores of vitamin Common causes (related to malabsorption) B12 are 3 to 5 mg, and the recommended adult Autoimmune gastritis (pernicious anemia) daily intake is 2.4 μg.7,8 Celiac disease Causes of vitamin B12 defi ciency are listed in Table 2. Dietary defi ciency of vitamin B12 Infl ammatory bowel disease occurs less frequently than folate defi ciency because body stores can last for years owing Surgical , gastric bypass, ileal resection to effi cient enterohepatic recycling mecha- Less common causes nisms. Although uncommon, dietary B12 defi ciency can occur even in industrialized Nutritional countries in strict vegans and vegetarians, or (strict vegans, breastfed infants of mothers with vitamin B12 defi ciency) in breastfed infants of mothers with vitamin Nitrous oxide abuse B defi ciency. 12 Diphyllobothrium latum infection Complex absorption pathway Pancreatic insuffi ciency Dietary absorption of vitamin B12 is a complex process that begins with (also Drug effect (metformin, proton pump inhibitors) known as transcobalamin I or R-binder) pro- Inherited disorders affecting intrinsic factor or the cubam receptor duction by the salivary glands. Rare inherited disorder When food is digested in the stomach by (eg, methylmalonic acidemia, transcobalamin II defi ciency) gastric acid and pepsin, free vitamin B12 is re- leased and binds to haptocorrin.4,9 Information from references 4, 5, and 7. Simultaneously, gastric parietal cells se- crete intrinsic factor, which cannot interact Pernicious anemia Macrocytic with the vitamin B12-haptocorrin complex. and autoimmune gastritis Not until food moves into the duodenum, Chronic atrophic autoimmune gastritis is an anemia where trypsin and other pancreatic enzymes autoimmune process directed specifi cally at ei- with a mean cleave haptocorrin, is vitamin B free to 12 ther gastric parietal cells or intrinsic factor, or corpuscular bind to intrinsic factor.9 The resultant vi- both.10–12 Parietal cell damage leads to reduced tamin B12-intrinsic factor complex binds to production of gastric acid and intrinsic factor, volume > 100 fL the cubam receptor on the mucosal surface accompanied by a compensatory increase in is the hallmark of enterocytes in the ileum. From there, vi- serum gastrin levels. Decreased intrinsic fac- of megalo- tamin B12 is transported into the circulation tor leads to signifi cantly reduced absorption by multidrug resistance protein 1, where it is of dietary vitamin B12, resulting in pernicious blastic anemia readily bound by its transport protein trans- anemia. cobalamin II.7,9 Chronic atrophic autoimmune gastritis The vitamin B12-transcobalamin complex affects the body and fundus of the stomach, then binds to the transcobalamin receptors replacing normal oxyntic mucosa with atro- on hematopoietic stem cells (and other cell phic-appearing mucosa, often with associated types), allowing uptake of the complex, with intestinal metaplasia.11 subsequent lysosomal degradation of transco- The associated infl ammatory infi ltrate con- balamin. Free vitamin B12 is then available for sists predominantly of lymphocytes and plasma cellular metabolism. cells. Enterochromaffi n-like cell is Nearly every step of this pathway can be also seen in biopsies of the fundus or stomach disrupted in various pathologic states, but lack body (highlighted by staining for chromo- of intrinsic factor secondary to pernicious ane- granin A and synaptophysin) and is thought to mia is the cause of vitamin B12 defi ciency in be a precursor to neuroendocrine (carcinoid) most cases. tumors. In addition to having vitamin B12 defi -

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CASE 1: An older man with suspected myelodysplastic syndrome

A 68-year-old man with no signifi cant past medi- and occasional giant metamyelocytes cal history presented from prison to the emergency and band forms department with fatigue, occasional shortness of • Mildly increased ring sideroblasts (10%) seen with breath, weight loss, and numbness and tingling of iron stain both hands. • Megakaryocyte dysplasia in the form of small hy- Initial complete blood cell count fi ndings showed polobated forms. pancytopenia with macrocytic anemia, with the fol- fi ndings of multilineage dysplasia, in lowing values: addition to megaloblastic changes, were strongly sug- • count 1.81 × 109/L (reference gestive of myelodysplastic syndrome. range 4.5–10) Further evaluation • 6.2 g/dL (14–18) Additional testing yielded the following results: • Mean corpuscular volume 121.5 fL (80–95) • Serum folate level 18.1 ng/mL (> 4.7) • count 41 × 109/L (150–450). • Serum vitamin B12 level < 150 pg/mL (232–1,245) Because of his clinical symptoms and severe pan- • Parietal cell antibody positive (1:40) cytopenia with macrocytosis, bone marrow biopsy was • Conventional cytogenetics: normal male karyo- performed to evaluate for myelodysplastic syndrome type and acute leukemia. • Hematologic neoplasm next-generation-sequenc- Bone marrow biopsy results ing panel (62 genes): negative for disease-associ- Findings from bone marrow aspirate smear and core ated mutations. biopsy included the following (Figure 1): In conjunction with normal cytogenetic and next- • Hypercellularity (70%–80%; reference range generation-sequencing panel results, undetectable 30%–70%) vitamin B12 levels helped confi rm severe vitamin B12 • Erythroid hyperplasia, indicated by a reduced ratio defi ciency. This may be the underlying cause of the of myeloid to erythroid precursor cells (0.7; refer- cytopenias and dysplasia. It was speculated that a re- ence range 2–4:1) and 2% blasts stricted diet during incarceration was the source of • Severe megaloblastic changes in the erythroid and the problem. granulocytic lineages; erythroid precursors showed Treatment signifi cant nuclear-cytoplasmic dyssynchrony, Intramuscular (1,000 μg) was multinucleation, nuclear budding, nuclear ir- started, followed by high-dose oral cyanocobalamin regularities, and ; granulocytic (1,000 μg/day). Abnormal complete blood cell count precursors showed hypersegmentation of mature fi ndings improved, as did neurologic symptoms.

ciency, patients with chronic atrophic autoim- vitamin leads to many similar manifestations. mune gastritis are at increased risk of gastric Both are involved in single carbon adenocarcinomas and neuroendocrine tumors. transfer (methylation), which is necessary for Hyperplasia of gastrin cells can be identi- the conversion of deoxyuridylate to deoxythy- 7 fi ed using gastrin immunohistochemistry on midylate. Insuffi cient folate or vitamin 12B gastric antral biopsies. Serologic testing for leads to decreased thymidine available for antiparietal and anti-intrinsic factor antibod- DNA synthesis, hampering cell division and ies, as well as increased serum levels of gastrin, replication. help confi rm the diagnosis.10–12 In synthesis, 5,10-methylene- tetrahydrofolate serves as the methyl donor,7 ■ FOLATE AND VITAMIN B12 METABOLISM after which it is converted to dihydrofolate, ARE INTERTWINED which must be reduced and then methylated

Folate and vitamin B12 metabolism are inti- to be used again. The reduction of dihydro- mately interconnected, so defi ciency in either folate to tetrahydrofolate by dihydrofolate re-

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Figure 1. A,B: Bone marrow aspirate smears showing severe megaloblastic changes: nuclear-cytoplasmic dyssynchrony, binucleation, nuclear irregularity, and basophilic stip- pling in erythroid lineage cells, and also hypersegmentation, nuclear-cytoplasmic dys- synchrony, and giant metamyelocytes or band forms in granulocytes (Wright-Giemsa, × 1,000). C: Bone marrow core biopsy showing hypercellularity, erythroid hyperplasia, left shift in maturation, and small dysplastic megakaryocytes (arrow) (hematoxylin and eosin, × 400). D: Small dysplastic megakaryocytes highlighted by CD61 immunohistochemistry on the core biopsy. E,F: Increased ring sideroblasts in iron stain on the aspirate smears.

ductase is targeted by multiple drugs,5,13 which • Chemotherapeutics, eg, analogues have the effect of decreasing available de- (fl udarabine, cladribine, and thiogua- oxythymidylate for DNA synthesis, resulting nine) in megaloblastic anemia. • Allopurinol, a xanthine oxidase inhibitor used to treat gout. ■ DRUG EFFECTS Drugs that affect pyrimidine synthesis in- clude13: Owing to vitamin fortifi cation of common • Immunomodulatory drugs, eg, lefl unomide foods in developed countries, megaloblastic and terifl unomide anemia related to vitamin defi ciency is increas- • Chemotherapeutics, eg, , gem- 2,14 ingly uncommon. However, this reduced in- citabine, and fl uorouracil cidence is offset by a growing list of drugs that • , an immunosuppressant and can cause megaloblastic anemia by interfering chemotherapeutic with DNA synthesis in various ways.2,4,13 • Sulfa drugs and . Drugs that affect purine synthesis in- Numerous drugs from multiple classes 2,13 clude : can reduce folate or vitamin B12 absorption, • Immunosuppressants, eg, and although this rarely leads to clinically signifi - mycophenolate mofetil cant defi ciency.

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CASE 2: A young woman with worsening anemia and family history of autoimmune disease

A young woman, age 17, presented to the emergency • Antiparietal cell antibody negative department with headache and abdominal pain that • Anti-intrinsic factor antibody positive. had worsened over the previous month. She had sought The laboratory and clinical fi ndings were consis-

medical care several times over the past 6 months with tent with vitamin B12 defi ciency, and the presence of similar symptoms, when moderate anemia was attrib- anti-intrinsic factor antibody confi rmed the diagnosis uted to iron defi ciency from heavy menses (the most of pernicious anemia. Although it tends to occur in common cause of anemia in women of reproductive older women, it is occasionally seen in young adults. age). Family history was notable for her sister having au- A strong family history of autoimmune disease is com- toimmune thyroid disease and type 1 diabetes mellitus. mon in patients with pernicious anemia. On additional questioning, she reported paresthesias in She was also tested for the following: the hands. Physical examination revealed decreased pro- • Serum thyroid-stimulating hormone level 6.72 prioception and vibratory sense and a wide-based gait. μU/mL (0.40–2.80) Results of initial testing were as follows: • Free thyroxine 1.3 ng/dL (0.8–1.5) • Hemoglobin 6.8 g/dL (down from 8.5 g/dL at her • Thyroid peroxidase antibody 1,224 IU/mL (< 5.6). last visit) These fi ndings indicate she is at risk for developing • Mean corpuscular volume 104.2 fL (elevated) symptomatic thyroid disease. • White blood cell count 6.91 × 109/L (normal) Treatment • Platelet count 300 × 109/L (normal) Treatment was started with parenteral cyanocobala- • Peripheral blood smear: several hypersegmented neu- min, at fi rst with daily intramuscular 1,000-μg cyano- trophils with no left-shift in maturation (Figure 2). cobalamin injections. Treatments were then weekly, Further tests were performed: then monthly, with rapid improvement of hemato- • Direct antiglobulin test negative logic symptoms and slower but complete resolution of • Serum iron, ferritin, and total iron-binding capac- her neurologic symptoms. ity normal Future considerations • Haptoglobin < 10 mg/dL (reference range 31–238) Given the personal and family history of autoimmune • 4,131 U/L (135–214) disease, a diagnosis of polyglandular autoimmune syn- • Relative reticulocytosis— count 48 × drome should be considered. Extensive clinical and 109/L (18–100); 2.6% (0.4%–2.0%). laboratory evaluation for other signs of autoimmune

• Serum vitamin B12 < 150 pg/mL (232–1,245) disease is warranted. Antiadrenal and GAD65 anti- • Serum folate normal body testing should be performed to assess risk for de- • Serum methylmalonic acid 8,361 nmol/L (79–376) veloping adrenal insuffi ciency.

■ CLINICAL FEATURES marrow that exhibit nuclear-to-cytoplasmic 7 Vitamin B defi ciency causes hematologic dyssynchrony. Ineffective erythropoiesis leads 12 to intramedullary hemolysis, classically with and neuropsychiatric manifestations that may high lactate dehydrogenase and undetectable occur together or independently.15,16 Mega- haptoglobin, but without schistocytes in the loblastic anemia due to folate defi ciency and peripheral blood. other causes shares the same hematologic Symptoms secondary to anemia include manifestations as vitamin B12 defi ciency but fatigue, shortness of breath, and poor exercise 4,7 lacks the neurologic features (see Case 2). tolerance. Hematologic features Neuropsychiatric features The most common hematologic manifestation Vitamin B12 defi ciency can cause subacute is megaloblastic anemia, which includes mac- combined degeneration of the dorsal and lat- rocytic erythrocytes in the peripheral blood eral columns of the spinal cord. Patients may and megaloblastic precursor cells in the bone experience bilateral and symmetrical pares-

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Figure 2. A,B: Two hypersegmented neutrophils (> 6 nuclear lobes) in a peripheral blood smear (Wright-Giemsa, × 1,000). thesia and decreased vibratory and positional ■ INITIAL EVALUATION sense. Psychiatric manifestations include While there is no gold standard for diagnos- memory loss, delirium, dementia, depression, ing megaloblastic anemia, appropriate clinical 15,17,18 mania, and hallucinations. and laboratory evaluation can usually estab- Diets lacking Atypical presentations lish the correct diagnosis. folate are rare Although neuropsychiatric symptoms often History and physical examination in countries develop after hematologic abnormalities, A complete history and physical examina- with vitamin more than 25% of patients with neurolog- tion are imperative. Targeted questions should 20 fortifi cation ic manifestations of vitamin B12 defi ciency cover the following areas : have either a normal hematocrit or a normal 17 • Diet—vegan or vegetarian? MCV. • Surgical history—gastric or ileal resection? Why certain patients are prone to hema- • Gastrointestinal symptoms—celiac disease tologic complications of vitamin defi ciency or gastritis? and other patients have neurologic sequelae • Neurologic symptoms such as paresthesias, remains unclear, but those with underlying numbness, ataxia, or gait disturbances? abnormalities such as pre-existing neurologic • Medications—folate antagonists, chemo- comorbidities or bone marrow failure condi- therapeutics? tions may be more likely to develop side ef- fects related to those conditions. Initial blood work The complete blood cell count reveals anemia Other fi ndings that is generally macrocytic (MCV > 100 fL). An increased risk of thrombosis is seen in Anemia can be seen in isolation or with leukope- vitamin B12 and folate defi ciency, possibly as nia or thrombocytopenia. Note that concurrent a consequence of hyperhomocysteinemia.19 iron defi ciency anemia can result in a normal Atrophic (swollen, erythematous, MCV but increased red cell distribution width. smooth tongue) is a common, albeit nonspe- The peripheral blood smear shows mor- cifi c, fi nding in vitamin B12 defi ciency. phologic changes in red blood cells (RBCs),

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including marked size variation () the other hand, pregnancy, drugs such as oral and abnormal morphology (), contraceptives and anticonvulsants, human including macro-ovalocytes, teardrop cells, immunodefi ciency virus infection, and folate

microcytes, and in severe cases, schistocytes, defi ciency can falsely reduce vitamin B12 levels. basophilic stippling, Howell-Jolly bodies, and For borderline cobalamin levels (200–400 nucleated RBCs. pg/mL), additional laboratory testing, includ- Polychromasia is not typically present. In ing serum methylmalonic acid and serum the setting of cytopenias and neurologic symp- homocysteine levels, should be performed.5 toms, absence of schistocytes excludes throm- Methylmalonic acid and homocysteine are

botic thrombocytopenic purpura. intermediaries in vitamin B12 metabolism Hypersegmented neutrophils (ie, ≥ 1% of and are increased in vitamin B12 defi ciency. neutrophils having 6 or more nuclear lobes, or Homocysteine is also elevated in folate defi - ≥ 5% of neutrophils with 5 nuclear lobes) in ciency and renal disease but methylmalonic the setting of macrocytic anemia are consid- acid is not, making it a more specifi c marker of ered specifi c for megaloblastic anemia and are 4 vitamin B12 defi ciency. rarely seen in other diseases.2,7 Vitamin B12 defi ciency secondary to in- Folate laboratory evaluation creased intramedullary destruction of RBC Laboratory testing for suspected folate defi - precursors can cause undetectable haptoglo- ciency starts with evaluating serum or plasma bin levels and elevated lactate dehydrogenase folate. Fasting serum folate generally refl ects and indirect bilirubin. tissue levels of folate; however, postprandial For suspected pernicious anemia, sero- increases in folate occur and can cause falsely logic testing for antiparietal cell and anti- normal results in nonfasting samples.6 After a intrinsic factor antibodies, as well as gastrin, 10 meal, increased serum folate occurs within 2 are useful. Antiparietal cell antibodies in hours, then quickly returns to baseline. Falsely patients with autoimmune pernicious ane- elevated folate levels can also be seen with mia demonstrate high sensitivity (81%) and sample hemolysis and vitamin B defi ciency. specifi city (90%), while anti-intrinsic factor Lack of 12 antibodies have high specifi city (100%) but In the latter situation, inadequate vitamin B12 intrinsic factor causes folate to be trapped in the 5-methyl- low sensitivity (27%–50%). The combina- secondary tetrahydrofolate state.5 tion of these 2 tests signifi cantly increases An alternative method of evaluating folate their diagnostic performance, with 73% sen- to pernicious stores is RBC folate, which refl ects the folate sitivity and 100% specifi city in pernicious anemia status of the prior 3 months and has the advan- anemia.23,24 Elevated gastrin is highly sensi- tive (85%) for pernicious anemia; however, is the cause tage of not being affected by recent dietary in- take. Disadvantages include slower turn-around it can also be elevated in Zollinger-Ellison of vitamin B12 time and higher cost. Also, recent transfusion syndrome, therapy with proton pump inhibi- defi ciency of RBCs can lead to inaccurate results, as it will tors or histamine 2 receptor blockers, Helico- refl ect the folate level of the donor. bacter pylori infection, or renal failure.4,24 in most cases Vitamin B12 laboratory evaluation ■ SPECIAL TESTING Specifi c laboratory evaluation for vitamin B12 defi ciency begins with total serum cobalamin Neuroimaging for atypical cases 21,22 levels. Vitamin B12 levels lower than 200 Neuroimaging is unnecessary for patients with pg/mL are highly suggestive of defi ciency, al- a classic clinical presentation of vitamin B12 though false-positive and false-negative results defi ciency. However, in suspected cases with- can happen. A normal cobalamin level makes out hematologic manifestations, magnetic defi ciency unlikely, although it may occur in resonance imaging is indicated. The most

nitrous oxide exposure or abuse, which in- consistent fi nding in vitamin B12 defi ciency is 7 volves metabolically inactive vitamin B12. In a symmetric, abnormally increased T2 signal addition, in pernicious anemia, anti-intrinsic intensity, involving the posterior or lateral factor antibodies can interfere with vitamin columns (or both) in the cervical and tho- 5 14 B12 assays, leading to falsely normal results. On racic spinal cord.

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Bone marrow aspiration and biopsy detailed clinical and medication history and

If vitamin defi ciency or drug effects cannot be laboratory fi ndings, including vitamin B12 and determined clinically and by laboratory test- folate levels, can help determine the correct ing as the cause of anemia, bone marrow bi- diagnosis. opsy may provide useful information. In meg- Megaloblastic anemia can also mimic ma- aloblastic anemia, the bone marrow shows the lignant conditions. Cytopenias, combined following: with severe megaloblastic fi ndings in the bone • Hypercellularity for age marrow, overlap with the neoplastic processes • Erythroid predominance, with a decreased of low-grade myelodysplastic syndrome or myeloid-to-erythroid ratio acute myeloid leukemia.3,25,26 Diagnostic con- • A left-shift in hematopoietic maturation. siderations include myelodysplastic syndrome Megaloblastic changes are best appreci- with excess blasts and erythroid predomi- ated with bone marrow aspirate smears using nance, as well as pure erythroid leukemia (ie, Wright-Giemsa stain. The typical fi ndings in a neoplastic proliferation of immature ery- the erythroid lineage include increased over- throid cells with > 80% erythroids and > 30% all size and nuclear-cytoplasmic dyssynchrony proerythroblasts) without increased myeloid (ie, a large, immature-appearing nucleus with blasts.27 an open chromatin pattern accompanied by Although myelodysplastic syndrome and a mature-appearing cytoplasm).7 Findings are severe megaloblastic anemia have overlap- also apparent in the granulocytic lineage, as ping features, careful morphologic evaluation seen by giant metamyelocytes and bands.7 Hy- of the bone marrow aspirate and biopsy can persegmented neutrophils can be seen in ei- identify differentiating characteristics. Dys- ther peripheral blood or bone marrow smears. plastic features characteristic of myelodysplas- Occasionally, megakaryocytes are also affect- tic syndrome that are not typical of megalo- ed, with large forms having hyperlobation and blastic anemia include the following: decreased cytoplasmic granularity. • Hyposegmentation or hypogranulation of In severe vitamin defi ciency, dysplastic granulocytes features can be observed, most often involv- • Hypolobation or small forms of megakaryo- Vitamin B12 ing the erythroid lineage in the form of nu- cytes defi ciency clear irregularities, eg, binucleation, multinu- • Hypogranular causes cleation, nuclear fragmentation, and nuclear • Increased blasts. budding, which resemble features seen in my- hematologic Laboratory fi ndings, including vitamin B12 elodysplastic syndrome (see “Differential di- and folate levels, conventional cytogenetics, and neuro- agnosis” below). and next-generation sequencing, can also Severe ineffective hematopoiesis can help distinguish the 2 entities.26 Identifying psychiatric markedly increase iron stores (detectable an acquired clonal abnormality, such as a my- manifestations with iron stain), although ring sideroblasts are elodysplastic syndrome-associated cytogenet- rarely seen in megaloblastic anemia. ic abnormality or mutation, would strongly Gastric biopsy support a neoplastic process. Gastric biopsy can confi rm chronic atrophic autoimmune gastritis. ■ TREAT UNDERLYING PROBLEM After establishing the diagnosis, treatment ■ DIFFERENTIAL DIAGNOSIS should be initiated promptly. Treatment is Establishing the correct diagnosis of megalo- specifi c to the underlying condition and usu- blastic anemia is paramount, as the treatment ally involves supplementing the defi cient and prognosis for different conditions can be vitamin. With either vitamin B12 or folate vastly different. The differential diagnosis supplementation, the rapid bone marrow re- includes conditions that cause nonmegalo- sponse can push borderline iron stores into blastic macrocytic anemia, such as medica- defi ciency, so patients should be monitored tion effects, ethanol abuse, hypothyroidism, for iron and provided with supplementation liver disease, and post-splenectomy status. A as needed. Megaloblastic anemia secondary

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TABLE 3

a Estimated cost of treatment per month for vitamin B12 and folate defi ciency Formulation Dose Cost per month

Vitamin B12 Intramuscular injection 1,000 μg/mL, single vial of 1 mL $5–$15 Oral 1,000 μg/pill, 30 pills per month $2–$5 Nasal spray 500 μg/spray, single spray per day, $500–$640 carton of 4 Sublingual lozenges 3,000 μg/lozenge, single lozenge per day, $5 ~ 30 lozenges per month Folic acid 1 mg/pill, 30 pills per month $3–$5

a The dose and cost are adapted from GoodRx.com.

to drug effect is best treated by stopping the Multiple supplementation options are causative agent if feasible. available, with the choice depending on clini- Generally, response to therapy is rapid, cal and nonclinical factors. All forms are gen- with hemoglobin levels improving within a erally well tolerated, but adverse reactions such 28,29 week. Neurologic symptoms of vitamin B12 as hypersensitivity have been reported. defi ciency generally resolve more slowly than hematologic symptoms and may not resolve Formulations vary completely. Vitamin B12 can be supplemented in different forms; noted preferences vary worldwide: cya- ■ FOLATE SUPPLEMENTATION nocobalamin in the United States, hydroxy- Hypersegment- cobalamin in Europe, and methylcobalamin in Megaloblastic anemia secondary to folate de- ed neutrophils Asia.30 Although all forms are well absorbed, fi ciency is generally treated with oral folate, hydroxycobalamin may be best for those with in the setting as it is most often caused by dietary defi ciency inherited errors of cobalamin metabolism. Cy- of macrocytic rather than malabsorption. For supplementa- anocobalamin is more expensive but appears tion and treatment, it is available as either of to be more stable for oral supplementation. anemia are con- the following: Vitamin B is available as a pill, sublingual • The synthetic form, known as folic acid or 12 sidered specifi c lozenge, intranasal spray, and intramuscular pteroylglutamic acid injection. Oral and intramuscular administra- for megalo- • The naturally occurring form, folinic acid. tion are the most widely studied and used. blastic anemia Folate defi ciency is typically treated with oral folic acid 1 to 5 mg per day.28 This dosage is Oral vs intramuscular vitamin B12 more than the recommended dietary allowance About 1.2% of oral cobalamin is passively ab- of 400 μg per day, thereby allowing for adequate sorbed unbound, while the remainder requires repletion even in the setting of malabsorption. intrinsic factor to be absorbed in the ileum.31 Treatment is continued for the duration of he- Eussen et al32 found that high-dose oral vita- matologic recovery or until the cause of defi - min B12 (> 200 × the recommended dietary ciency is addressed. In patients with malabsorp- allowance of 2.4 μg/day) produces adequate tion, treatment is continued indefi nitely. reductions in methylmalonic acid. However, despite multiple studies demonstrating the ef- ■ VITAMIN B12 SUPPLEMENTATION fectiveness of oral vitamin B12 even in perni- Prompt treatment is particularly important for cious anemia, a 2018 Cochrane review33 found

patients with vitamin B12 defi ciency in order a lack of data demonstrating equivalence to to prevent neurologic symptoms from becom- intramuscular administration, mainly due to a ing permanent. limited number of quality randomized studies.

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The most common oral dosage is 1,000 to severity of neurologic symptoms at diagnosis 2,000 μg daily, compared with 1,000 μg intra- may be predictive of outcome.3,36 muscularly daily for 7 days, then weekly for a Serum vitamin B12 levels fl uctuate signifi - month, then monthly thereafter.34 cantly with the timing of oral or intramuscular Advantages of intramuscular administra- dosing, making testing of little value except in tion include improved adherence and less- diagnosis. Serum methylmalonic acid levels do frequent dosing during the monthly mainte- not necessarily correlate well with clinical im- nance stage of treatment. As intramuscular provement, as patients sometimes continue to administration avoids reliance on gastrointes- report symptoms after levels have normalized. tinal tract absorption, it is particularly useful Therefore, a combination of clinical and labora- in patients who have undergone bowel sur- tory testing is used to monitor therapy response. geries or in patients with severe neurologic Laboratory testing should include com- impairments who need optimal and quick re- plete blood cell and reticulocyte counts. The pletion of vitamin B12. Unless the patient self- reticulocyte count should increase after ap- administers it, the main disadvantages are the proximately 2 to 3 days, peaking at 5 to 7 inconvenience and increased costs associated days.37 We recommend checking a complete with receiving it at a medical facility. Actual blood cell count and reticulocyte count 4 monthly costs of oral and intramuscular for- weeks after the initiation of vitamin B12 ther- mulations are otherwise similar (Table 3).35 apy. The time point will also give an opportu- In general, mild vitamin B12 defi ciency nity to reassess the symptoms and plan a tran- should be treated with oral dosing, reserving sition to less-frequent dosing, if the response intramuscular dosing for patients with signifi - is adequate. cant neurologic symptoms, adherence issues, Hemoglobin typically starts increasing in a or extensive gastric or bowel resections. Pa- week, with expected complete normalization in tients with neurologic symptoms should have 4 to 8 weeks.37 Delayed or incomplete response frequent injections until neurologic symptoms should prompt further evaluation for other have disappeared and undergo more extended causes of anemia, including iron defi ciency. In Testing treatment if symptoms are severe. their dose-fi nding study, Eussen et al32 reported for suspected Intranasal absolute reductions of serum methylmalonic acid concentrations of at least 0.22 μmol/L at Given the variable absorption of intranasal folate supplementation, closer clinical and serum initial testing at 8 weeks and also at 16 weeks. defi ciency Although the expected reduction of methyl- methylmalonic acid monitoring is indicated starts with to ensure therapeutic response. If the response malonic acid level is not standardized to vita- min B dosage, evidence nevertheless supports is inadequate, switching to the intramuscular 12 serum or monitoring methylmalonic acid levels to assess route should be considered. plasma folate response to B12 supplementation, especially in Monitoring patients with pernicious anemia.32,37 We rec- There is no standard approach to monitoring ommend doing this at 4 weeks after initiation response. Symptoms of anemia usually im- and on follow-up every 6 months to a year, as prove fairly quickly, but neurologic symptoms long as the complete blood cell count remains tend to resolve slowly or incompletely. The normal and there are no new symptoms. ■

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