1 INTRODUCTION

Megaloblastic anemia (MA) in the form of autoimmune pernicious anemia (PA) was described by physicians as early as the 1800s. A case report described a patient with numbness of the upper extremities, large red blood cells, atrophic gastric mucosa, and hypercellular bone marrow with increased erythroblasts.1 Whipple, Minot, and Murphy earned a Nobel Prize in 1934 for the discovery of a treatment for PA.1, 2 In the 1950s, the complete structure of cobalamin was discovered as well as the biochemical pathway detailing the conversion of homocysteine to methionine.1 Despite remarkable progress in our understanding of autoimmune pernicious anemia, this diagnosis can be uniquely challenging, a feature that has even been reported in the lay press. For example, the Wall Street Journal published in an article titled “Vitamin B12 Deficiency: The Serious Health Problem That's Easy To Miss” in April 2019, recognizing its frequency and its sometimes diagnostically elusive nature.3 Herein, we review the classic features of MA, describe uniquely challenging scenarios from the blood and bone marrow perspective with illustrative cases, and discuss the diagnostic traps and pitfalls encountered during the workup of MA, specific laboratory testing, and limitations thereof.

1.1 Overview

Megaloblastic anemia, a subtype of macrocytic anemia, frequently develops secondary to vitamin B12 (cobalamin) or much less often, folate deficiency. DNA synthesis is dependent on these coenzymes, and deficiencies in these can cause ineffective DNA synthesis and, thus, ineffective hematopoiesis. RNA and protein synthesis, however, are not as reliant on these coenzymes; subsequently, nuclear-cytoplasmic maturational dyssynchrony occurs, giving rise to giantism of proliferating cells and cells with delayed nuclear maturation. Ineffective hematopoiesis results from intramedullary cell death and eventually peripheral cytopenias, despite a hypercellular bone marrow.2, 4, 5 While vitamin B12 and folate deficiencies are primarily responsible for acquired MA, drug-induced MA is becoming increasingly recognized, as a number of medications biochemically alter DNA synthesis.6-8

Macrocytosis of red blood cells (RBC) in adults is defined as a mean corpuscular volume (MCV) of greater than 100 fL, and literature reports a prevalence as high as 3.6% in the general adult population,6, 9 though a significant percentage of these is not necessarily associated with an anemia.9 Complete blood count (CBC) data provide essential information about the presence of macrocytosis, the degree of anemia, and other features. However, MA does not always present with macrocytosis.10 While the exact prevalence of MA is unknown, it is not insignificant, and the distribution in various populations has shifted over time, particularly for folate deficiency, with one review noting a range of 0.06% to 1.6% prevalence of folate deficiency across multiple studies.11 Racial and ethnic differences in folate deficiency prevalence were also reported in a recent United States-based study, potentially reflecting differences in social determinants of health.11 Although still more prevalent in developing countries where malnutrition remains problematic, the incidence of folate deficiency-related MA has decreased, particularly among elderly and pregnant individuals, due to vitamin supplementation and food fortification. Prevalence of vitamin B12 deficiency is population dependent, with prevalence in the United Kingdom and the United states of around 6% in individuals younger than 60 years but closer to 20% in older individuals.12 There is significantly greater prevalence in certain subpopulations of African and Asian countries, in particular Kenyan schoolchildren (70%) and Indian preschool children (80%) and Indian adults (80%) in one study.12 While the most common cause of vitamin B12 deficiency is autoimmune atrophic gastritis (PA), dietary deficiencies and other causes of malabsorption are common. Autoimmune atrophic gastritis shows a predilection for females and those of northern European or African descent. It affects all age groups; however, it usually occurs in individuals older than 40 years with a mean age range of 59–62 years.5, 13

The combined efforts of clinicians, pathologists, and laboratory professionals are essential to establish a diagnosis of acquired MA, and integration of clinical features, hematologic findings, laboratory studies, and morphologic examination is key. A thorough history and physical examination often reveal characteristic symptoms and signs including fatigue, jaundice, pallor, and atrophy of mucosal surfaces. Neurologic manifestations may arise such as neuropathy and neuropsychiatric disorders including depression and forgetfulness. Patient history should include a full accounting of prior surgeries and list of medications. A CBC is essential with inclusion of RBC indices, and morphologic assessment of a peripheral blood smear review for “clues” for MA is essential. Serum vitamin B12 and folate concentrations should be assessed in all cases of possible MA, though these values should be interpreted with caution given limitations in sensitivity and specificity for deficiency states, particularly for vitamin B12. Additional studies of potential diagnostic utility include thyroid and liver function studies, serum and storage iron, methylmalonic acid (MMA), total homocysteine, and anti-intrinsic factor (IF) and anti-parietal cell antibody assays. Bone marrow evaluation in the setting of MA, though usually unnecessary, may be needed when hematologic and clinical parameters are particularly concerning for an underlying hematologic neoplasm or other disorder.2, 7

However, acquired MA cases do not always present “classically,” requiring heightened awareness by both hematologists and pathologists. Patients may present with severe neurologic features and an elevated lactate dehydrogenase (LDH) with fragmented RBCs, concerning for thrombotic thrombocytopenic purpura (TTP),14, 15 or may have severe pancytopenia with bone marrows showing hypercellularity and morphologic atypia, raising concern for myelodysplastic syndrome (MDS) or even acute leukemia.16 Consequently, the diagnosis of MA can be missed in some cases, as features can be quite complex and heterogeneous, and vitamin B12 concentrations may not factor into initial workups given nonspecific symptomatology. In addition, issues may arise in laboratory testing including false normal cobalamin concentrations, adding to this diagnostic challenge.5, 17, 18

2 CLASSIC FEATURES OF MEGALOBLASTIC ANEMIA 2.1 Vitamin B12 and folate

Vitamin B12 is synthesized by certain bacterial species including those in human gut flora, and dietary sources are almost exclusively of animal origin. Consequently, strict vegetarians, vegans, and even exclusively breastfed infants of mothers with vitamin B12 deficiency are at risk for developing deficiency due to insufficient dietary intake. Other etiologies include malabsorption, increased demand, defective transport, and disorders of metabolism2 with a list of causes in Table 1.

TABLE 1. Causes of vitamin B12 deficiency and folate deficiency Vitamin B12 deficiency Malabsorption Issues Pernicious anemia (autoimmune atrophic gastritis)a Gastrectomy, total or partial Bariatric surgery/gastric bypass Ileal resection Inherited disorders affecting vitamin B12 absorption Mild chronic atrophic gastritis Inflammatory bowel disease Pancreatic insufficiency Nitrous oxide abuse Metformin, proton pump inhibitors Fish tapeworm infestation Inadequate Intake Strict vegans or vegetarians Breastfeeding infants of vitamin B12-deficient mothers Decreased intake of dairy and meat products Drugs Interference of Vitamin B12 absorption (aminosalicylic acid, isoniazid, colchicine, neomycin, and metformin) Interference of folate absorption (aminosalicylic acid, birth control pills, estrogens, tetracyclines, penicillins, phenytoin, phenobarbital, chloroquine, and primaquine) Interference of pyrimidine synthesis (hydroxyurea, methotrexate, gemcitabine, mercaptopurine, fluorouracil, trimethoprim, and leflunomide) Folate analog activity (methotrexate, pemetrexed, and trimethoprim) Recreational abuse of nitrous oxide Others Pregnancyb Lactationb Folate deficiency Inadequate intake Alcoholism Drug abuse Overcooked foods Depressed, elderly, and individuals of lower socioeconomic backgrounds Malabsorption issues Celiac disease (sprue) Inflammatory bowel disease Short bowel syndrome, surgical or functional Malignancy Increased requirement Pregnancy Lactation Infants Malignancy Chronic hemolytic anemia Drugs Interference of folate absorption (oral contraceptives, estrogens, antibiotics, and aminosalicylic acid) Interference of pyrimidine synthesis (antineoplastic agents and immunomodulators)

The physiology of B12 absorption is complex. Haptocorrin, produced by the salivary glands, binds and protects the acid-sensitive B12 (consumed within food) in the stomach. Pancreatic enzymes then digest haptocorrin, releasing B12 and allowing it to bind to IF secreted by gastric parietal cells, forming the vitamin B12-IF complex. This complex then binds to the cubam receptor in the terminal ileum and becomes internalized; IF undergoes lysosomal degradation, and vitamin B12 is transported into the blood by multidrug resistance protein 1. It then binds the delivery protein, transcobalamin, rendering vitamin B12 available for cellular uptake.2, 5, 19 This pathway may be disrupted at a number of different steps, but the loss of IF due to autoantibody production, as seen in PA, is the most common.2, 5

Folate is produced by microorganisms and plants, which account for dietary sources including vegetables and fruits. The most common cause of folate deficiency is poor dietary intake, as seen in alcoholics, drug abusers, elderly, and individuals of lower socioeconomic status; however, folate deficiency is now rare due to fortification of foods in many countries. An increased folate requirement is also a common cause as seen in pregnancy and other settings. Absorption defects at the level of the jejunum (surgical resection and celiac sprue) and metabolic disorders can also be implicated.2, 7, 20, 21 A list of causes is provided in Table 1.

Vitamin B12 and folate are intricately related, crucial for DNA synthesis, and are vital for cell division of all proliferating cells, and particularly in this context, hematopoiesis. A deficiency in either vitamin will impair DNA synthesis; this results in cellular arrest at the S phase of the cell cycle, leading to replication errors and ultimately apoptosis.2, 22 The biochemical pathway is illustrated in Figure 1.

Metabolic pathway of vitamin B12 and folate

2.2 Vitamin B12 deficiency and pernicious anemia

Pernicious anemia, or autoimmune atrophic gastritis, is the most common cause of severe vitamin B12 deficiency. The gastric parietal cells are essentially atrophied, thereby decreasing IF production and subsequently preventing B12 absorption.5, 23 Like other autoimmune diseases, autoantibodies play a role in PA, with up to 60% and 90% of patients having antibodies against IF and gastric parietal cell antigens, respectively.23 The presence of anti-IF antibodies is highly specific but relatively insensitive for PA.24 Conversely, anti-parietal cell antibodies are sensitive, reported in up to 90% of patients with PA, but nonspecific, as they are also present in other autoimmune disorders and in some healthy individuals..25

Clinically, patients may present with symptoms of anemia, including pallor, fatigue, dyspnea, and tachycardia. Manifestations also include mucosal atrophy of the tongue and gastrointestinal (GI) tract, causing glossitis and secondary malabsorption and anorexia, respectively.5, 23 Neuropsychiatric manifestations in PA are well-described. Defective myelin synthesis occurs initially, and axonal disruption and/or degeneration and neuronal death in the spinal cord follow. This results in paresthesias and weakness with subsequent ataxia and paralysis. If untreated, PA can result in central nervous system compromise, with the so-called megaloblastic madness that can include depression, mental changes, paranoia, delusions, and frank psychosis.2, 5, 23 Of note, neuropsychiatric symptoms can be seen in the absence of hematologic abnormalities, and the hematologic and neuropsychiatric effects can develop independently of one another.

The classic hematologic features of vitamin B12 deficiency include the following: macrocytic normochromic anemia (MCV >100 fL) with anisopoikilocytosis, macro-ovalocytes, and even schistocytes, Howell-Jolly bodies, and basophilic stippling in severe cases; a low reticulocyte count and markedly increased red cell distribution width (RDW); mild leukopenia and thrombocytopenia; and hypersegmented neutrophils (defined as ≥1% of neutrophils having 6 or more nuclear lobes or ≥5% of neutrophils with 5 nuclear lobes). However, these features may be masked in some patients with concurrent iron deficiency, thalassemia, or other chronic disorders which can normalize the MCV (although the RDW is likely still significantly increased). Moreover, neurologic impairment occurs in approximately 25% of cases without an elevated MCV.11, 18, 22, 26 Increased LDH, increased indirect bilirubin, and low haptoglobin can also be seen as a result of ineffective erythropoiesis from intramedullary hemolysis as well as peripheral destruction, as these large macrocytes undergo damage as they pass through the spleen.2, 23 The characteristic antibody findings are as mentioned above, and serum gastrin is elevated with a sensitivity of 85%.5 Additional laboratory tests in PA reveal the following: decreased serum vitamin B12, normal or increased folate, increased MMA, and elevated homocysteine. Refer to Table 2 for classic laboratory features of vitamin B12 deficiency.

TABLE 2. Classic laboratory features of vitamin B12 and folate deficiencies Test Vitamin B12 deficiency Folate deficiency Comments CBC and peripheral blood smear

Macrocytic anemia (MCV >100 fL)

Mild leukopenia and thrombocytopenia

Low reticulocyte count

Hypersegmented neutrophils

Macrocytic anemia (MCV >100 fL)

Mild leukopenia and thrombocytopenia

Low reticulocyte count

Hypersegmented neutrophils

Concurrent iron deficiency, thalassemia, and other conditions can result in normal MCV Vitamin B12 Decreased in anemias secondary to vitamin B12 deficiency and PA Normal or decreased in severe folate deficiency Interference by autoantibodies including intrinsic factor antibodies Folate Normal or increased in PA Decreased in folate deficiency Concentrations fluctuate with diet RBC folate Decreased in vitamin B12 deficiency Decreased in folate deficiency RBC folate concentrations reflect folate status at time of cell formation Methylmalonic acid Increased in vitamin B12 deficiency Normal in folate deficiency More specific than serum vitamin B12 (increased in impaired renal function); equal sensitivity to serum vitamin B12 Total homocysteine Increased in vitamin B12 deficiency Increased in vitamin B12 deficiency

Poor specificity (elevated in renal failure, alcohol abuse, hypothyroidism, inborn errors of homocysteine metabolism)

More sensitivea

Interpretation requires expertiseb

Holotranscobalamin Decreased in vitamin B12 deficiency Normal in folate deficiency

More reliable indicator of vitamin B12 status; not clinically validated in the United States

See commentc

Lactate dehydrogenase Markedly elevated in megaloblastic anemia Markedly elevated in megaloblastic anemia Due to intramedullary destruction of cells; results can be falsely elevated by hemolysis Indirect bilirubin Mildly elevated in megaloblastic anemia Mildly elevated in megaloblastic anemia Due to intramedullary destruction and hemolysis of abnormal RBCs Haptoglobin Decreased in megaloblastic anemia Decreased in megaloblastic anemia Due to hemolysis resulting in free hemoglobin Intrinsic factor antibodies Present in 50%–70% of cases of PA Negative Very specific for PA Parietal cell antibodies Present in 90% of cases Negative Sensitive for PA but seen in other disorders Abbreviations: CBC, Complete Blood Count; MCV, Mean Corpuscular Volume; PA, Pernicious Anemia; RBC, Red blood cells.

Though not usually indicated in this setting, a bone marrow evaluation would demonstrate hypercellularity and erythroid and granulocytic hyperplasia with megaloblastic maturation (Figure 2A,B). In the erythroid lineage, nuclear maturation lags behind that of the cytoplasm, leading to a dyssynchronous nuclear-cytoplasmic pattern.2, 5, 23 This pattern in the myeloid lineage results in giantism of bands and metamyelocytes and hypersegmentation of neutrophils. Megakaryocytic changes have also been described with large, hyperlobated forms. Despite bone marrow hypercellularity, hematopoiesis is ultimately ineffective, leading to cytopenias.2, 5, 22, 23 Refer to Table 3 for characteristic bone marrow findings.

Bone marrow aspirate smear illustrates an increase in early erythroid precursors (A) with dyssynchronous nuclear-cytoplasmic maturation. This is often accompanied by megaloblastic changes in the (B) myeloid series with giant bands (black arrow) and hypersegmented neutrophils (red arrow). (C) Megaloblastic anemia in patient with normal mean corpuscular volume (MCV) of 93 fL. Red blood cells show marked anisopoikilocytosis with oval macrocytes, with hypersegmented neutrophil present. (D) Megaloblastic anemia in patient presenting with pancytopenia due to vitamin B12 deficiency. The blood smear image shows a single circulating erythroid precursor with absolute neutrophil count 0.9 × 109/μl, hemoglobin 7.8 g/dL, and platelet count 48 000/μl. Note oval macrocytes

TABLE 3. Prototypic peripheral blood, CBC, and bone marrow findings in megaloblastic anemia Peripheral blood and CBC data Macrocytic anemia (>100 fL) Low reticulocyte count Mild leukopenia Mild thrombocytopenia Hypersegmented neutrophils Bone marrow Hypercellularity Megaloblastic changes in erythroid lineage Erythroid hyperplasia Increased pronormoblasts Nuclear-cytoplasmic asynchrony (finely dispersed nuclei and mature cytoplasm with hemoglobinization) Binucleated and multinucleated forms Nuclear budding Fragmented nuclei Megaloblastic changes in granulocytic lineage Giantism of bands and metamyelocytes Nuclear hypersegmentation of mature granulocytes 2.3 Folate deficiency

Patients with folate deficiency also present with symptoms of anemia. Patients with GI illnesses or prior GI surgical procedures are at risk for either B12 or folate deficiency. However, folate deficiency is clearly more prevalent in patients with alcoholism or drug dependency (see Table 1). Neurologic changes are not classically associated with folate deficiency, though they have rarely been reported in this clinical setting.27 Folate deficiency during embryogenesis can cause neural tube defects, the incidence of which has decreased significantly due to improved supplementation and consumption.7, 21 Considering the time course of anemia development may also be helpful in clinically differentiating vitamin B12 and folate deficiency. In adults, folate stores may become exhausted after only 3–5 months, whereas vitamin B12 stores are more longstanding and thus a B12 deficiency may manifest after two to five years.2, 7, 28

The hematologic findings of folate deficiency are similar to vitamin B12 deficiency. Laboratory testing of folate and vitamin B12 is required; in folate deficiency, folate will be decreased, and vitamin B12 can be normal or moderately decreased in the setting of severe folate deficiency.2, 7 While both vitamin B12 and folate are required for the metabolism of homocysteine to methionine, only vitamin B12 is involved in the metabolism of methylmalonyl-coenzyme A to succinyl-coenzyme A. Hence, an isolated folate deficiency will produce an elevated homocysteine concentration and a normal concentration of MMA.4, 7 If bone marrow biopsy and aspirate are performed, the findings will be comparable to those seen in vitamin B12 deficiency. Refer to Tables 2 and 3 for classic laboratory features of folate deficiency and bone marrow findings in MA.

2.4 Drug-induced megaloblastic anemia

Drug-induced MA is becoming increasingly recognized.8 Many drugs that are frequently used in clinical practice, such as antimicrobials, antineoplastic agents, antiepileptic agents, and immunomodulators, are responsible for altering the biochemical process of DNA synthesis by interfering with the cellular availability of vitamin B12 and folate through various mechanisms. One major pathway in drug-induced megaloblastic anemia is the interference of thymidine synthesis, which is highly dependent on folate and Vitamin B12.8 The broad list of drug mechanisms of action includes: modulation of purine metabolism; pyrimidine synthesis interference; inhibition of folate or vitamin B12 absorption; folate analog activity; promotion of vitamin B12 excretion; and destruction of vitamin B12.6-8 Drug-induced MA can be classically seen in patients on certain medications such as methotrexate for immunosuppression, hydroxyurea for cytoreductive therapy, or certain antibiotic, antiepileptic, or chemotherapeutic agents. Additional examples are included in Table 1.

3 UNIQUE FEATURES OF MEGALOBLASTIC ANEMIA AND DIAGNOSTIC TRAPS 3.1 Normal mean corpuscular volume

Macrocytosis is a common clinical finding in MA and can precede the anemia by months; however, increased MCV (>100 fL) is not always present in patients with MA. Iron deficiency, thalassemia, chronic illnesses (causing active inflammatory states), or renal disease may normalize the MCV in patients with MA or even produce a microcytic picture.11 If the clinician dismisses MA as a possibility based solely on a non-elevated MCV, patients with MA masked by another comorbidity may remain undiagnosed and potentially suffer from sequelae of this disease. One study showed that 33% of patients lacked macrocytosis.29 Review of the peripheral blood smear may show a dimorphic population of red cells with microcytes and macro-ovalocytes, marked anisocytosis, and hypersegmented neutrophils (Figure 2C). Diagnosticians must be cognizant of concurrent conditions that can obscure the typical findings, like macrocytosis, that are often, but not always, seen in MA. However, other clues to this diagnosis are evident on peripheral blood smear review.

3.2 Pancytopenia

Recent studies report that pancytopenia is present in 5% of patients with pernicious anemia, while earlier reports describe a higher incidence of this potentially life-threatening manifestation in MA patients.16, 30, 31 Although rare, pregnant women with folate deficiency can also present with pancytopenia.31 Careful review of the blood smear for clues to MA is essential. MA manifesting as pancytopenia can be confirmed without bone marrow evaluation if it is suspected, making clinical suspicion and review of the peripheral blood smear of utmost importance. In addition to an elevated RDW, morphologic features on peripheral blood smear include oval macrocytes, hypersegmented neutrophils, and circulating nucleated RBCs, if present, may show the classic sieve-like chromatin (Figure 2D).

3.3 Features concerning for myeloid neoplasms

Bone marrow morphology in patients with MA can be misleading, mimicking a myeloid neoplasm ranging from myelodysplastic syndrome (MDS) to acute myeloid leukemia, notably pure erythroid leukemia (PEL), especially in the setting of pancytopenia. Marked bone marrow hypercellularity with florid erythroid lineage expansion, markedly increased erythroblasts with megaloblastic changes, and nonspecific “stress”-related dysplasia can all be misinterpreted as features of a myeloid neoplasm32 (Figure 3A). Furthermore, MDS can manifest with predominant megaloblastic dysplasia mimicking MA. Bastida et al. developed a four-step approach to diagnose a “hidden MDS” in patients with anemia of unknown etiology and/or macrocytosis, with the fourth step being bone marrow evaluation. This approach allowed them to identify patients not only with MDS, but other causes of anemia, including MA.16

(A) Bone marrow aspirate smear shows a preponderance of erythroblasts with deeply basophilic cytoplasm and megaloblastic chromatin, raising concern for acute erythroid leukemia or a high-grade myelodysplastic syndrome given erythroid predominance and the presence of precursors with irregular nuclei (arrows). (B) Striking features of microangiopathy in peripheral blood smear of patient with megaloblastic anemia, presenting with marked anemia (hemoglobin 4.7 g/dL), mean corpuscular volume 122 fL, and thrombocytopenia (platelets 27 000/μl), accompanied by severe anisopoikilocytosis with macro-ovalocytes, red blood cell fragments, and increased polychromasia. (C) Megaloblastic anemia in seven-month old infant with failure to thrive, with bone marrow aspirate smear showing megaloblastic erythroid and myeloid precursors. The patient presented with macrocytic anemia with hemoglobin 4.0 g/dL and platelet count 40 000/μl. Image courtesy of Dr. Carl R. Kjeldsberg, MD. (D) Megaloblastic anemia in 43-year old patient with partially treated vitamin B12 deficiency. She presented previously with mild macrocytic anemia, was found to have vitamin B12 deficiency, and was on 5 days of repletion therapy at time of this biopsy. Megaloblastic changes appear more persistent in the myeloid series with frequent giant bands (arrows) than in erythroid precursors

Erroneous diagnosis of MA as neoplasia has been well-documented in the medical literature. Konda et al. reported a case of pancytopenia, hemolysis, and low reticulocyte count with the initial bone marrow showing increased immature cells.33 A repeat marrow evaluation showed hypercellularity with trilineage hematopoiesis, numerous giant band forms, left-shifted erythroid hyperplasia, and atypia in all three lineages. Flow cytometry and cytogenetics were normal. Parenteral vitamin B12 was administered with resolution of all laboratory counts. Uncommonly, transient cytogenetic abnormalities can be present in MA, further complicating diagnosis. Parmentier et al. described a case of severe pancytopenia, marked hemolysis, dysplastic bone marrow findings with suspicion for increased blasts, and an aberrant granulocytic antigen pattern by flow cytometry with abnormal cytogenetics.34 The patient was ultimately diagnosed with PA and started on vitamin B12 supplementation; the repeat bone marrow performed 2 weeks after hematologic recovery showed persistent megaloblastic atypia without increased blasts, and with resolution of the cytogenetic and flow cytometric aberrations.34 Singh et al. also report a case that was originally classified as MDS with excess blasts-1 (MDS-EB-1).35 Repeat bone marrow evaluation reportedly revealed marked erythroid dysplasia and hyperplasia with the presence of hypersegmented neutrophils and no increase in blasts. Vitamin B12 and folate concentrations were both low, supplementation was initiated, and follow-up studies revealed improvement in previously abnormal laboratory parameters.35 A literature review in 2017 identified 14 other case reports from the 1950s to present, which included 15 patients total, of vitamin B12 deficiency with associated transient chromosomal aberrations, many of which resolved with replacement vitamin B12 therapy.36

Cases of macrocytic anemia may be sent for analysis by next-generation sequencing to evaluate the possibility of myeloid neoplasm. Somatic mutations can be detected by these panels in the absence of neoplasia in conditions like clonal cytopenias of undetermined significance (CCUS). Mutations identified in MA patients should not be used as presumptive evidence of neoplasia in the absence of other diagnostic findings and where B12 and folate deficiency have not been ruled out.

Nonneoplastic and neoplastic bone marrow findings often overlap, and immunohistochemistry (IHC) can be a useful tool in distinguishing the two. A 2012 study evaluated the sensitivity and specificity of CD34-positive megakaryocytes in nonneoplastic and neoplastic bone marrows. The authors found that ≥30% CD34 reactivity of megakaryocytes was associated with MA and MDS, which can be used to distinguish t