case study megaloblastic anemia

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• Case Study: Vitamin B12 Deficiency

Volume 35 Number 4 | August 2021

Minh kosfeld, phd, mlt(ascp) cm, patient physical examination on admission.

Our patient is a previously healthy 56-year-old white man who presents with progressive neuropathy and declining mental status over several months. The patient initially developed numbness of his fingertips and the balls of his feet and began to lose motor control of his hands, which manifested as dropping objects or flinging them as he tried to pick them up. As symptoms progressed, he had visual tracking problems that were severe enough to interfere with driving a car, and he developed short-term memory loss and slowing of cognitive function.

Laboratory Results

Diagnosis and Treatment

Based on the clinical presentation and laboratory findings, a diagnosis of psychomotor regression due to B12 deficiency was made. The patient was then treated with a series of B12 intramuscular injections, which resulted in rapid remission of associated neurological symptoms. In follow-up laboratory examinations, his B12 level was normalized in two months (606 pg/mL), and MMA in eight months (197 nmol/L).

B12 is the largest and most complex of the water-soluble B vitamins. Since it contains cobalt, compounds with B12 activity are collectively called “cobalamins.” For humans, the only natural dietary sources of B12 are animal products (meat, dairy), in which it is bound to protein. The B12 absorption mechanism is complex, requiring several transporter proteins. First, it must be freed from the food matrix by gastric HCl and pepsin. Once liberated, it is transported to the duodenum by haptocorrin (transcobalamin I), a cobalamin-binding protein produced in the saliva. In the duodenum, pancreatic digestive enzymes free B12 from haptocorrin, allowing it to bind intrinsic factor (IF), a transporter protein synthesized by the parietal cells of the gastric mucosa. This B12-IF complex travels to the terminal ileum where it is absorbed and the B12 is separated from IF. B12 then is delivered to peripheral tissues and the liver by transcobalamin II and haptocorrin, respectively. 1,2

B12 is essential for DNA synthesis, hematopoiesis, and myelination. In different metabolically active forms, it functions as a cofactor for two different enzymes. As methylcobalamin, B12 activates cytoplasmic methionine synthase to convert homocysteine to the essential amino acid methionine. Methionine is required for the formation of S-adenosylmethionine, a universal methyl donor for almost 100 different substrates, including DNA, RNA, proteins, and lipids. As adenosylcobalamin, B12 activates the mitochondrial L-methylmalonyl-CoA mutase to convert L-methylmalonyl-CoA to succinyl-CoA in the metabolism of propionate, a short-chain fatty acid. 3,4 In B12 deficiency, these cofactors are unavailable, causing homocysteine and MMA to accumulate.

Despite our understanding of the metabolic disturbances resulting from B12 deficiency, its pathogenesis is not as well understood. Deficiency of B12 can lead to two major clinical syndromes, megaloblastic anemia and neuropathy. 1,3,4 It is thought that severe B12 deficiency interferes with the synthesis of DNA needed for hematopoiesis, leading to megaloblastic anemia, the appearance of hypersegmented neutrophils, and possible pancytopenia. 5 Regarding neuropathy, it is thought that long-term B12 deficiency may lead to impaired synthesis of ethanolamine, phospholipids, and sphingomyelin, resulting in altered myelin integrity. 6 An enigmatic feature of B12 deficiency is that its clinical presentations vary and may entail only hematologic or neurological abnormalities, or both. 1

Common diagnostic lab tests for B12 deficiency typically begin with serum B12 measurement and a complete blood count (CBC). Low B12 levels and evidence of megaloblastic anemia (decreased RBC, Hgb, Hct, WBC, platelet count, increased MCV, large oval RBCs and hypersegmented neutrophils) indicate B12 deficiency. For ambiguous results, MMA and homocysteine levels should also be measured to confirm. 2,4 Serum MMA is the most sensitive marker of B12 status where its increase indicates decreased tissue B12. However, it also rises with renal insufficiency and tends to be higher in older adults. Plasma homocysteine is a sensitive indicator for early B12 deficiency where it rises quickly as B12 status declines. However, it also rises in folate or B6 deficiency and especially with renal insufficiency, reducing its specificity. 3,4 Additional tests include serum or RBC folate to differentiate causes of macrocytic anemia, and Anti-IF or Anti-parietal cell antibodies to confirm pernicious anemia. 1,2

Caregivers should be aware of the different clinical presentations of B12 deficiency and screen for it in high-risk populations. Risk factors include insufficient dietary B12 intake (vegetarians), lack of intrinsic factor (autoimmune pernicious anemia), food-bound malabsorption (atrophic gastritis of aging or chronic H. pylori infection), gastrointestinal surgery (post gastrectomy or ileal resection), pancreatic or intestinal disorders (chronic pancreatitis, Crohn’s, or Celiac disease), genetic disorders (Transcobalamin II deficiency), and use of certain drugs (long-term use of metformin, H2 receptor antagonists or Proton-pump inhibitors). 1,2,3

Diagnosis of B12 deficiency can be complicated since symptoms may be vague and lab test results can be equivocal. Despite the low B12, high MMA, and significant neurologic symptoms, this patient’s homocysteine level was normal, and he was not anemic. With no known risk factor for B12 deficiency, assays were done for anti-IF antibodies to evaluate for pernicious anemia and AChR binding antibodies for Myasthenia Gravis. However, this patient’s positive symptomatic and serologic responses to B12 supplementation suggest that simple vitamin B12 deficiency was the etiology. Fortunately, B12 therapy reversed his most severe neurological symptoms, although that may not always be the case. Since the reason for the deficiency is unknown, he will continue to receive B12 supplementation indefinitely.

• Michael J Shipton and Jecko Thachil, Vitamin B12 deficiency–A 21st century perspective. Clin Med (Lond). 2015 Apr; 15(2): 145–150. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4953733/
• https://labtestsonline.org/conditions/vitamin-b12-and-folate-deficiencies
• Vitamin B12. https://ods.od.nih.gov/factsheets/VitaminB12-HealthProfessional/
• Bishnu Prasad Devkota. Methylmalonic Acid. https://emedicine.medscape.com/article/2108967-overview#showall
• Mark J Koury, Prem Ponka. New insights into erythropoiesis: the roles of folate, vitamin B12, and iron. Annu Rev Nutr. 2004;24:105-31. https://pubmed.ncbi.nlm.nih.gov/15189115/
• Brahim El Hasbaoui, Nadia Mebrouk, Salahiddine Saghir, Abdelhkim El Yajouri, Rachid Abilkassem, and Aomar Agadr. Vitamin B12 deficiency: case report and review of literature. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8140678/

Minh Kosfeld is Director/Assistant Professor, Investigative and Medical Sciences Program, in the Department of Clinical Health Sciences at Saint Louis University in Missouri.

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Severe megaloblastic anemia: Vitamin deficiency and other causes

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Megaloblastic anemia causes macrocytic anemia from ineffective red blood cell production and intramedullary hemolysis. The most common causes are folate (vitamin B 9 ) deficiency and cobalamin (vitamin B 12 ) deficiency. Megaloblastic anemia can be diagnosed based on characteristic morphologic and laboratory findings. However, other benign and neoplastic diseases need to be considered, particularly in severe cases. Therapy involves treating the underlying cause—eg, with vitamin supplementation in cases of deficiency, or with discontinuation of a suspected medication.

The hallmark of megaloblastic anemia is macrocytic anemia (mean corpuscular volume > 100 fL), often associated with other cytopenias.

Dysplastic features may be present and can be difficult to differentiate from myelodysplastic syndrome.

Megaloblastic anemia is most commonly caused by folate deficiency from dietary deficiency, alcoholism, or malabsorption syndromes or by vitamin By deficiency, usually due to pernicious anemia.

Both vitamin deficiencies cause hematologic signs and symptoms of anemia; vitamin B 12 deficiency also causes neurologic symptoms.

Oral supplementation is available for both vitamin deficiencies; intramuscular vitamin B 12 supplementation should be used in cases involving severe neurologic symptoms or gastric or bowel resection.

Not all megaloblastic anemias result from vitamin deficiency, but most do. Determining the underlying cause and initiating prompt treatment are critical, as prognosis and management differ among the various conditions.

This article describes the pathobiology, presentation, evaluation, and treatment of severe megaloblastic anemia and its 2 most common causes: folate (vitamin B 9 ) and cobalamin (vitamin B 12 ) deficiency, with 2 representative case studies.

• MEGALOBLASTIC ANEMIA OVERVIEW

Megaloblastic anemia is caused by defective DNA synthesis involving hematopoietic precursors, resulting in ineffective red blood cell production (erythropoiesis) and intramedullary hemolysis. Macrocytic anemia with increased mean corpuscular volume (MCV), defined as more than 100 fL, is the hallmark of megaloblastic anemia, but leukopenia and thrombocytopenia are also frequently present.

The incidence of macrocytosis is as high as 4% in the general population, but megaloblastic anemia accounts for only a small fraction. 1 Nonmegaloblastic causes of macrocytic anemia include ethanol abuse, myelodysplastic syndrome, aplastic anemia, hypothyroidism, liver disease, and drugs. 2 , 3 Although these causes are associated with increased MCV, they do not lead to the other features of megaloblastic anemia.

The most frequent causes of megaloblastic anemia are deficiencies of vitamin B 9 (folate) or vitamin B 12 (cobalamin) ( Table 1 ). Lessfrequent causes include congenital disorders (inborn errors of metabolism), drugs (particularly chemotherapeutics and folate antagonists), micronutrient deficiencies, and nitrous oxide exposure. 4 , 5

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Characteristics of vitamin B 12 vs folate deficiency

• FOLATE DEFICIENCY

Folate is found in green leafy vegetables, fruits, nuts, eggs, and meats. Normal body stores of folate are 5 to 30 mg. The recommended daily allowance depends on age, sex, and pregnancy status, but is generally 400 μg in adults and 600 μg during pregnancy. 6

Folate deficiency has 3 main causes 4 , 5 :

Reduced intake from diets lacking folate (rare in countries with vitamin fortification) and alcoholism (see Case 1 )

Decreased absorption from disorders affecting nutrient absorption in the small bowel, eg, celiac disease, inflammatory bowel disease, and tropical sprue

Increased demand from pregnancy, hemolytic anemia, puberty, and eczematous conditions.

• VITAMIN B 12 DEFICIENCY

Vitamin B 12 is produced by microorganisms and is found almost exclusively in foods of animal origin. Normal body stores of vitamin B 12 are 3 to 5 mg, and the recommended adult daily intake is 2.4 μg. 7 , 8

Causes of vitamin B 12 deficiency are listed in Table 2 . Dietary deficiency of vitamin В 12 occurs less frequently than folate deficiency because body stores can last for years owing to efficient enterohepatic recycling mechanisms. Although uncommon, dietary B 12 deficiency can occur even in industrialized countries in strict vegans and vegetarians, or in breastfed infants of mothers with vitamin B 12 deficiency.

Causes of vitamin B 12 deficiency

Complex absorption pathway

Dietary absorption of vitamin B 12 is a complex process that begins with haptocorrin (also known as transcobalamin I or R-binder) production by the salivary glands.

When food is digested in the stomach by gastric acid and pepsin, free vitamin B 12 is released and binds to haptocorrin. 4 , 9

Simultaneously, gastric parietal cells secrete intrinsic factor, which cannot interact with the vitamin B 12 -haptocorrin complex. Not until food moves into the duodenum, where trypsin and other pancreatic enzymes cleave haptocorrin, is vitamin B 12 free to bind to intrinsic factor. 9 The resultant vitamin B 12 -intrinsic factor complex binds to the cubam receptor on the mucosal surface of enterocytes in the ileum. From there, vitamin B 12 is transported into the circulation by multidrug resistance protein 1, where it is readily bound by its transport protein transcobalamin II. 7 , 9

The vitamin B 12 -transcobalamin complex then binds to the transcobalamin receptors on hematopoietic stem cells (and other cell types), allowing uptake of the complex, with subsequent lysosomal degradation of transcobalamin. Free vitamin B 12 is then available for cellular metabolism.

Nearly every step of this pathway can be disrupted in various pathologic states, but lack of intrinsic factor secondary to pernicious anemia is the cause of vitamin B 12 deficiency in most cases.

Pernicious anemia and autoimmune gastritis

Chronic atrophic autoimmune gastritis is an autoimmune process directed specifically at either gastric parietal cells or intrinsic factor, or both. 10 – 12 Parietal cell damage leads to reduced production of gastric acid and intrinsic factor, accompanied by a compensatory increase in serum gastrin levels. Decreased intrinsic factor leads to significantly reduced absorption of dietary vitamin B 12 , resulting in pernicious anemia.

Chronic atrophic autoimmune gastritis affects the body and fundus of the stomach, replacing normal oxyntic mucosa with atrophic-appearing mucosa, often with associated intestinal metaplasia. 11

The associated inflammatory infiltrate consists predominantly of lymphocytes and plasma cells. Enterochromaffin-like cell hyperplasia is also seen in biopsies of the fundus or stomach body (highlighted by staining for chromogranin A and synaptophysin) and is thought to be a precursor to neuroendocrine (carcinoid) tumors. In addition to having vitamin B 12 deficiency, patients with chronic atrophic autoimmune gastritis are at increased risk of gastric adenocarcinomas and neuroendocrine tumors.

CASE 1: An older man with suspected myelodysplastic syndrome

A 68-year-old man with no significant past medical history presented from prison to the emergency department with fatigue, occasional shortness of breath, weight loss, and numbness and tingling of both hands.

Initial complete blood cell count findings showed pancytopenia with macrocytic anemia, with the following values:

White blood cell count 1.81 × 10 9 /L (reference range 4.5-10)

Hemoglobin 6.2 g/dL (14-18)

Mean corpuscular volume 121.5 fL (80-95)

Platelet count 41 × 10 9 /L (150-450).

Because of his clinical symptoms and severe pancytopenia with macrocytosis, bone marrow biopsy was performed to evaluate for myelodysplastic syndrome and acute leukemia.

Bone marrow biopsy results

Findings from bone marrow aspirate smear and core biopsy included the following ( Figure 1 ):

Hypercellularity (70%-80%; reference range 30%-70%)

Erythroid hyperplasia, indicated by a reduced ratio of myeloid to erythroid precursor cells (0.7; reference range 2-4:1) and 2% blasts

Severe megaloblastic changes in the erythroid and granulocytic lineages; erythroid precursors showed significant nuclear-cytoplasmic dyssynchrony, multinucleation, nuclear budding, nuclear irregularities, and basophilic stippling; granulocytic precursors showed hypersegmentation of mature neutrophils and occasional giant metamyelocytes and band forms

Mildly increased ring sideroblasts (10%) seen with iron stain

Megakaryocyte dysplasia in the form of small hypolobated forms.

Bone marrow findings of multilineage dysplasia, in addition to megaloblastic changes, were strongly suggestive of myelodysplastic syndrome.

Further evaluation

Additional testing yielded the following results:

Serum folate level 18.1 ng/mL (> 4.7)

Serum vitamin B 12 level < 150 μg/mL (232-1, 245)

Parietal cell antibody positive (1:40)

Conventional cytogenetics: normal male karyotype

Hematologic neoplasm next-generation-sequencing panel (62 genes): negative for disease-associated mutations.

In conjunction with normal cytogenetic and nextgeneration-sequencing panel results, undetectable vitamin B 12 levels helped confirm severe vitamin B 12 deficiency. This may be the underlying cause of the cytopenias and dysplasia. It was speculated that a restricted diet during incarceration was the source of the problem.

Intramuscular cyanocobalamin (1, 000 μg) was started, followed by high-dose oral cyanocobalamin (1, 000 μg/day). Abnormal complete blood cell count findings improved, as did neurologic symptoms.

Hyperplasia of gastrin cells can be identified using gastrin immunohistochemistry on gastric antral biopsies. Serologic testing for antiparietal and anti-intrinsic factor antibodies, as well as increased serum levels of gastrin, help confirm the diagnosis. 10 – 12

• FOLATE AND VITAMIN B 12 METABOLISM ARE INTERTWINED

Folate and vitamin В 12 metabolism are intimately interconnected, so deficiency in either vitamin leads to many similar manifestations. Both vitamins are involved in single carbon transfer (methylation), which is necessary for the conversion of deoxyuridylate to deoxythymidylate. 7 Insufficient folate or vitamin B 12 leads to decreased thymidine available for DNA synthesis, hampering cell division and replication.

In pyrimidine synthesis, 5, 10-methylenetetrahydrofolate serves as the methyl donor, 7 after which it is converted to dihydrofolate, which must be reduced and then methylated to be used again. The reduction of dihydrofolate to tetrahydrofolate by dihydrofolate reductase is targeted by multiple drugs, 5 , 13 which have the effect of decreasing available deoxythymidylate for DNA synthesis, resulting in megaloblastic anemia.

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A, B : Bone marrow aspirate smears showing severe megaloblastic changes: nuclear-cytoplasmic dyssynchrony, binucleation, nuclear irregularity, and basophilic stippling in erythroid lineage cells, and also hypersegmentation, nuclear-cytoplasmic dyssynchrony, 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.

• DRUG EFFECTS

Owing to vitamin fortification of common foods in developed countries, megaloblastic anemia related to vitamin deficiency is increasingly uncommon. 2 , 14 However, this reduced incidence is offset by a growing list of drugs that can cause megaloblastic anemia by interfering with DNA synthesis in various ways. 2 , 4 , 13

Drugs that affect purine synthesis include 2 , 13 :

Immunosuppressants, eg, azathioprine and mycophenolate mofetil

Chemotherapeutics, eg, purine analogues (fludarabine, cladribine, and thioguanine)

Allopurinol, a xanthine oxidase inhibitor used to treat gout.

Drugs that affect pyrimidine synthesis include 13 :

Immunomodulatory drugs, eg, leflunomide and teriflunomide

Chemotherapeutics, eg, cytarabine, gemcitabine, and fluorouracil

Methotrexate, an immunosuppressant and chemotherapeutic

Sulfa drugs and trimethoprim.

Numerous drugs from multiple classes can reduce folate or vitamin B 12 absorption, although this rarely leads to clinically significant deficiency.

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

A young woman, age 17, presented to the emergency department with headache and abdominal pain that had worsened over the previous month. She had sought medical care several times over the past 6 months with similar symptoms, when moderate anemia was attributed to iron deficiency from heavy menses (the most common cause of anemia in women of reproductive age). Family history was notable for her sister having autoimmune thyroid disease and type 1 diabetes mellitus. On additional questioning, she reported paresthesias in the hands. Physical examination revealed decreased proprioception and vibratory sense and a wide-based gait.

Results of initial testing were as follows:

Hemoglobin 6.8 g/dL (down from 8.5 g/dL at her last visit)

Mean corpuscular volume 104.2 fL (elevated)

White blood cell count 6.91 × 10 9 /L (normal)

Platelet count 300 × 10 9 /L (normal)

Peripheral blood smear: several hypersegmented neutrophils with no left-shift in maturation ( Figure 2 ). Further tests were performed:

Direct antiglobulin test negative

Serum iron, ferritin, and total iron-binding capacity normal

Haptoglobin < 10 mg/dL (reference range 31-238)

Lactate dehydrogenase 4, 131 U/L (135-214)

Relative reticulocytosis—reticulocyte count 48 × 10 9 /L (18-100); 2.6% (0.4%-2.0%).

Serum vitamin B 12 < 150 μg/mL (232-1, 245)

Serum folate normal

Serum methylmalonic acid 8, 361 nmol/L (79-376)

Antiparietal cell antibody negative

Anti-intrinsic factor antibody positive.

A, B: Two hypersegmented neutrophils (> 6 nuclear lobes) in a peripheral blood smear (Wright-Giemsa, × 1, 000).

The laboratory and clinical findings were consistent with vitamin B 12 deficiency, and the presence of anti-intrinsic factor antibody confirmed the diagnosis of pernicious anemia. Although it tends to occur in older women, it is occasionally seen in young adults. A strong family history of autoimmune disease is common in patients with pernicious anemia.

She was also tested for the following:

Serum thyroid-stimulating hormone level 6.72 pU/mL (0.40-2.80)

Free thyroxine 1.3 ng/dL (0.8-1.5)

Thyroid peroxidase antibody 1, 224 IU/mL (< 5.6). These findings indicate she is at risk for developing symptomatic thyroid disease.

Treatment was started with parenteral cyanocobalamin, at first with daily intramuscular 1, 000-μg cyanocobalamin injections. Treatments were then weekly, then monthly, with rapid improvement of hematologic symptoms and slower but complete resolution of her neurologic symptoms.

Future considerations

Given the personal and family history of autoimmune disease, a diagnosis of polyglandular autoimmune syndrome should be considered. Extensive clinical and laboratory evaluation for other signs of autoimmune disease is warranted. Antiadrenal and GAD65 antibody testing should be performed to assess risk for developing adrenal insufficiency.

• CLINICAL FEATURES

Vitamin B 12 deficiency causes hematologic and neuropsychiatric manifestations that may occur together or independently. 15 , 16 Megaloblastic anemia due to folate deficiency and other causes shares the same hematologic manifestations as vitamin B 12 deficiency but lacks the neurologic features (see Case 2 ). 4 , 7

Hematologic features

The most common hematologic manifestation is megaloblastic anemia, which includes macrocytic erythrocytes in the peripheral blood and megaloblastic precursor cells in the bone marrow that exhibit nuclear-to-cytoplasmic dyssynchrony. 7 Ineffective erythropoiesis leads to intramedullary hemolysis, classically with high lactate dehydrogenase and undetectable haptoglobin, but without schistocytes in the peripheral blood.

Symptoms secondary to anemia include fatigue, shortness of breath, and poor exercise tolerance.

Neuropsychiatric features

Vitamin B 12 deficiency can cause subacute combined degeneration of the dorsal and lateral columns of the spinal cord. Patients may experience bilateral and symmetrical paresthesia and decreased vibratory and positional sense. Psychiatric manifestations include memory loss, delirium, dementia, depression, mania, and hallucinations. 15 , 17 , 18

Atypical presentations

Although neuropsychiatric symptoms often develop after hematologic abnormalities, more than 25% of patients with neurologic manifestations of vitamin B 12 deficiency have either a normal hematocrit or a normal MCV. 17

Why certain patients are prone to hematologic complications of vitamin deficiency and other patients have neurologic sequelae remains unclear, but those with underlying abnormalities such as pre-existing neurologic comorbidities or bone marrow failure conditions may be more likely to develop side effects related to those conditions.

Other findings

An increased risk of thrombosis is seen in vitamin B 12 and folate deficiency, possibly as a consequence of hyperhomocysteinemia. 19 Atrophic glossitis (swollen, erythematous, smooth tongue) is a common, albeit nonspecific, finding in vitamin B 12 deficiency.

• INITIAL EVALUATION

While there is no gold standard for diagnosing megaloblastic anemia, appropriate clinical and laboratory evaluation can usually establish the correct diagnosis.

History and physical examination

A complete history and physical examination are imperative. Targeted questions should cover the following areas 20 :

Diet—vegan or vegetarian?

Surgical history—gastric or ileal resection?

Gastrointestinal symptoms—celiac disease or gastritis?

Neurologic symptoms such as paresthesias, numbness, ataxia, or gait disturbances?

Medications—folate antagonists, chemotherapeutics?

Initial blood work

The complete blood cell count reveals anemia that is generally macrocytic (MCV > 100 fL). Anemia can be seen in isolation or with leukopenia or thrombocytopenia. Note that concurrent iron deficiency anemia can result in a normal MCV but increased red cell distribution width.

The peripheral blood smear shows morphologic changes in red blood cells (RBCs), including marked size variation (anisocytosis) and abnormal morphology (poikilocytosis), including macro-ovalocytes, teardrop cells, microcytes, and in severe cases, schistocytes, basophilic stippling, Howell-Jolly bodies, and nucleated RBCs.

Polychromasia is not typically present. In the setting of cytopenias and neurologic symptoms, absence of schistocytes excludes thrombotic thrombocytopenic purpura.

Hypersegmented neutrophils (ie, > 1% of neutrophils having 6 or more nuclear lobes, or > 5% of neutrophils with 5 nuclear lobes) in the setting of macrocytic anemia are considered specific for megaloblastic anemia and are rarely seen in other diseases. 2 , 7

Folate laboratory evaluation

Laboratory testing for suspected folate deficiency starts with evaluating serum or plasma folate. Fasting serum folate generally reflects tissue levels of folate; however, postprandial increases in folate occur and can cause falsely normal results in nonfasting samples. 6 After a meal, increased serum folate occurs within 2 hours, then quickly returns to baseline. Falsely elevated folate levels can also be seen with sample hemolysis and vitamin B 12 deficiency. In the latter situation, inadequate vitamin В 12 causes folate to be trapped in the 5-methyltetrahydrofolate state. 5

An alternative method of evaluating folate stores is RBC folate, which reflects the folate status of the prior 3 months and has the advantage of not being affected by recent dietary intake. Disadvantages include slower turn-around time and higher cost. Also, recent transfusion of RBCs can lead to inaccurate results, as it will reflect the folate level of the donor.

Vitamin B 12 laboratory evaluation

Specific laboratory evaluation for vitamin В 12 deficiency begins with total serum cobalamin levels. 21 , 22 Vitamin В 12 levels lower than 200 μg/mL are highly suggestive of deficiency, although false-positive and false-negative results can happen. A normal cobalamin level makes deficiency unlikely, although it may occur in nitrous oxide exposure or abuse, which involves metabolically inactive vitamin В 12 . 7 In addition, in pernicious anemia, anti-intrinsic factor antibodies can interfere with vitamin B 12 assays, leading to falsely normal results. 5 On the other hand, pregnancy, drugs such as oral contraceptives and anticonvulsants, human immunodeficiency virus infection, and folate deficiency can falsely reduce vitamin В 12 levels.

For borderline cobalamin levels (200-400 μg/mL), additional laboratory testing, including serum methylmalonic acid and serum homocysteine levels, should be performed. 5 Methylmalonic acid and homocysteine are intermediaries in vitamin В 12 metabolism and are increased in vitamin В 12 deficiency. Homocysteine is also elevated in folate deficiency and renal disease but methylmalonic acid is not, making it a more specific marker of vitamin В 12 deficiency. 4

Vitamin В 12 deficiency secondary to increased intramedullary destruction of REC precursors can cause undetectable haptoglobin levels and elevated lactate dehydrogenase and indirect bilirubin.

For suspected pernicious anemia, serologic testing for antiparietal cell and antiintrinsic factor antibodies, as well as gastrin, are useful. 10 Antiparietal cell antibodies in patients with autoimmune pernicious anemia demonstrate high sensitivity (81%) and specificity (90%), while anti-intrinsic factor antibodies have high specificity (100%) but low sensitivity (27%-50%). The combination of these 2 tests significantly increases their diagnostic performance, with 73% sensitivity and 100% specificity in pernicious anemia. 23 , 24 Elevated gastrin is highly sensitive (85%) for pernicious anemia; however, it can also be elevated in Zollinger-Ellison syndrome, therapy with proton pump inhibitors or histamine 2 receptor blockers, Helicobacter pylori infection, or renal failure. 4 , 24

• SPECIAL TESTING

Neuroimaging for atypical cases

Neuroimaging is unnecessary for patients with a classic clinical presentation of vitamin В 12 deficiency. However, in suspected cases without hematologic manifestations, magnetic resonance imaging is indicated. The most consistent finding in vitamin В 12 deficiency is a symmetric, abnormally increased T2 signal intensity, involving the posterior or lateral columns (or both) in the cervical and thoracic spinal cord. 14

Bone marrow aspiration and biopsy

If vitamin deficiency or drug effects cannot be determined clinically and by laboratory testing as the cause of anemia, bone marrow biopsy may provide useful information. In megaloblastic anemia, the bone marrow shows the following:

Hypercellularity for age

Erythroid predominance, with a decreased myeloid-to-erythroid ratio

A left-shift in hematopoietic maturation.

Megaloblastic changes are best appreciated with bone marrow aspirate smears using Wright-Giemsa stain. The typical findings in the erythroid lineage include increased overall size and nuclear-cytoplasmic dyssynchrony (ie, a large, immature-appearing nucleus with an open chromatin pattern accompanied by a mature-appearing cytoplasm). 7 Findings are also apparent in the granulocytic lineage, as seen by giant metamyelocytes and bands. 7 Hypersegmented neutrophils can be seen in either peripheral blood or bone marrow smears. Occasionally, megakaryocytes are also affected, with large forms having hyperlobation and decreased cytoplasmic granularity.

In severe vitamin deficiency, dysplastic features can be observed, most often involving the erythroid lineage in the form of nuclear irregularities, eg, binucleation, multinucleation, nuclear fragmentation, and nuclear budding, which resemble features seen in myelodysplastic syndrome (see “Differential diagnosis” below).

Severe ineffective hematopoiesis can markedly increase iron stores (detectable with iron stain), although ring sideroblasts are rarely seen in megaloblastic anemia.

Gastric biopsy

Gastric biopsy can confirm chronic atrophic autoimmune gastritis.

• DIFFERENTIAL DIAGNOSIS

Establishing the correct diagnosis of megaloblastic anemia is paramount, as the treatment and prognosis for different conditions can be vastly different. The differential diagnosis includes conditions that cause nonmegaloblastic macrocytic anemia, such as medication effects, ethanol abuse, hypothyroidism, liver disease, and post-splenectomy status. A detailed clinical and medication history and laboratory findings, including vitamin B 12 and folate levels, can help determine the correct diagnosis.

Megaloblastic anemia can also mimic malignant conditions. Cytopenias, combined with severe megaloblastic findings in the bone marrow, overlap with the neoplastic processes of low-grade myelodysplastic syndrome or acute myeloid leukemia. 3 , 25 , 26 Diagnostic considerations include myelodysplastic syndrome with excess blasts and erythroid predominance, as well as pure erythroid leukemia (ie, a neoplastic proliferation of immature erythroid cells with > 80% erythroids and > 30% proerythroblasts) without increased myeloid blasts. 27

Although myelodysplastic syndrome and severe megaloblastic anemia have overlapping features, careful morphologic evaluation of the bone marrow aspirate and biopsy can identify differentiating characteristics. Dysplastic features characteristic of myelodysplastic syndrome that are not typical of megaloblastic anemia include the following:

Hyposegmentation or hypogranulation of granulocytes

Hypolobation or small forms of megakaryocytes

Hypogranular platelets

Increased blasts.

Laboratory findings, including vitamin B 12 and folate levels, conventional cytogenetics, and next-generation sequencing, can also help distinguish the 2 entities. 26 Identifying an acquired clonal abnormality, such as a myelodysplastic syndrome-associated cytogenetic abnormality or mutation, would strongly support a neoplastic process.

• TREAT UNDERLYING PROBLEM

After establishing the diagnosis, treatment should be initiated promptly. Treatment is specific to the underlying condition and usually involves supplementing the deficient vitamin. With either vitamin B 12 or folate supplementation, the rapid bone marrow response can push borderline iron stores into deficiency, so patients should be monitored for iron and provided with supplementation as needed. Megaloblastic anemia secondary to drug effect is best treated by stopping the causative agent if feasible.

Generally, response to therapy is rapid, with hemoglobin levels improving within a week. Neurologic symptoms of vitamin B 12 deficiency generally resolve more slowly than hematologic symptoms and may not resolve completely.

• FOLATE SUPPLEMENTATION

Megaloblastic anemia secondary to folate deficiency is generally treated with oral folate, as it is most often caused by dietary deficiency rather than malabsorption. For supplementation and treatment, it is available as either of the following:

The synthetic form, known as folic acid or pteroylglutamic acid

The naturally occurring form, folinic acid.

Folate deficiency is typically treated with oral folic acid 1 to 5 mg per day. 28 This dosage is more than the recommended dietary allowance of 400 μg per day, thereby allowing for adequate repletion even in the setting of malabsorption. Treatment is continued for the duration of hematologic recovery or until the cause of deficiency is addressed. In patients with malabsorption, treatment is continued indefinitely.

• VITAMIN B 12 SUPPLEMENTATION

Prompt treatment is particularly important for patients with vitamin B 12 deficiency in order to prevent neurologic symptoms from becoming permanent.

Multiple supplementation options are available, with the choice depending on clinical and nonclinical factors. All forms are generally well tolerated, but adverse reactions such as hypersensitivity have been reported. 28 , 29

Formulations vary

Vitamin B 12 can be supplemented in different forms; noted preferences vary worldwide: cyanocobalamin in the United States, hydroxycobalamin in Europe, and methylcobalamin in Asia. 30 Although all forms are well absorbed, hydroxycobalamin may be best for those with inherited errors of cobalamin metabolism. Cyanocobalamin is more expensive but appears to be more stable for oral supplementation.

Vitamin B 12 is available as a pill, sublingual lozenge, intranasal spray, and intramuscular injection. Oral and intramuscular administration are the most widely studied and used.

Oral vs intramuscular vitamin B 12

About 1.2% of oral cobalamin is passively absorbed unbound, while the remainder requires intrinsic factor to be absorbed in the ileum. 31 Eussen et al 32 found that high-dose oral vitamin B 12 (> 200 × the recommended dietary allowance of 2.4 μg/day) produces adequate reductions in methylmalonic acid. However, despite multiple studies demonstrating the effectiveness of oral vitamin B 12 even in pernicious anemia, a 2018 Cochrane review 33 found a lack of data demonstrating equivalence to intramuscular administration, mainly due to a limited number of quality randomized studies.

The most common oral dosage is 1, 000 to 2, 000 μg daily, compared with 1, 000 μg intramuscularly daily for 7 days, then weekly for a month, then monthly thereafter. 34

Advantages of intramuscular administration include improved adherence and lessfrequent dosing during the monthly maintenance stage of treatment. As intramuscular administration avoids reliance on gastrointestinal tract absorption, it is particularly useful in patients who have undergone bowel surgeries or in patients with severe neurologic impairments who need optimal and quick repletion of vitamin В 12 Unless the patient selfadministers it, the main disadvantages are the inconvenience and increased costs associated with receiving it at a medical facility. Actual monthly costs of oral and intramuscular formulations are otherwise similar ( Table 3 ). 35

Estimated cost of treatment per month for vitamin B 12 and folate deficiency a

In general, mild vitamin B 12 deficiency should be treated with oral dosing, reserving intramuscular dosing for patients with significant neurologic symptoms, adherence issues, or extensive gastric or bowel resections. Patients with neurologic symptoms should have frequent injections until neurologic symptoms have disappeared and undergo more extended treatment if symptoms are severe.

Given the variable absorption of intranasal supplementation, closer clinical and serum methylmalonic acid monitoring is indicated to ensure therapeutic response. If the response is inadequate, switching to the intramuscular route should be considered.

There is no standard approach to monitoring response. Symptoms of anemia usually improve fairly quickly, but neurologic symptoms tend to resolve slowly or incompletely. The severity of neurologic symptoms at diagnosis may be predictive of outcome. 3 , 36

Serum vitamin В 12 levels fluctuate significantly with the timing of oral or intramuscular dosing, making testing of little value except in diagnosis. Serum methylmalonic acid levels do not necessarily correlate well with clinical improvement, as patients sometimes continue to report symptoms after levels have normalized. Therefore, a combination of clinical and laboratory testing is used to monitor therapy response.

Laboratory testing should include complete blood cell and reticulocyte counts. The reticulocyte count should increase after approximately 2 to 3 days, peaking at 5 to 7 days. 37 We recommend checking a complete blood cell count and reticulocyte count 4 weeks after the initiation of vitamin В 12 therapy. The time point will also give an opportunity to reassess the symptoms and plan a transition to less-frequent dosing, if the response is adequate.

Hemoglobin typically starts increasing in a week, with expected complete normalization in 4 to 8 weeks. 37 Delayed or incomplete response should prompt further evaluation for other causes of anemia, including iron deficiency. In their dose-finding study, Eussen et al 32 reported absolute reductions of serum methylmalonic acid concentrations of at least 0.22 pmol/L at initial testing at 8 weeks and also at 16 weeks. Although the expected reduction of methylmalonic acid level is not standardized to vitamin В 12 dosage, evidence nevertheless supports monitoring methylmalonic acid levels to assess response to В 12 supplementation, especially in patients with pernicious anemia. 32 , 37 We recommend doing this at 4 weeks after initiation and on follow-up every 6 months to a year, as long as the complete blood cell count remains normal and there are no new symptoms.

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• Heinila K ,
• Sillanaukee P ,
• Aslinia F ,
• Steensma DP
• Datta Mitra A
• Sobczynska-Malefora A ,
• Harrington DJ
• Institute of Medicine (US) Standing Committee on the Scientific Evaluation of Dietary Reference Intakes and its Panel on Folate, other B Vitamins, and Choline
• Schjonsby H
• Bizzaro N ,
• Farinati F ,
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ROBERT C. LANGAN, MD, FAAFP, AND ANDREW J. GOODBRED, MD

Am Fam Physician. 2017;96(6):384-389

Author disclosure: No relevant financial affiliations.

Vitamin B 12 deficiency is a common cause of megaloblastic anemia, various neuropsychiatric symptoms, and other clinical manifestations. Screening average-risk adults for vitamin B 12 deficiency is not recommended. Screening may be warranted in patients with one or more risk factors, such as gastric or small intestine resections, inflammatory bowel disease, use of metformin for more than four months, use of proton pump inhibitors or histamine H 2 blockers for more than 12 months, vegans or strict vegetarians, and adults older than 75 years. Initial laboratory assessment should include a complete blood count and serum vitamin B 12 level. Measurement of serum methylmalonic acid should be used to confirm deficiency in asymptomatic high-risk patients with low-normal levels of vitamin B 12 . Oral administration of high-dose vitamin B 12 (1 to 2 mg daily) is as effective as intramuscular administration for correcting anemia and neurologic symptoms. Intramuscular therapy leads to more rapid improvement and should be considered in patients with severe deficiency or severe neurologic symptoms. Absorption rates improve with supplementation; therefore, patients older than 50 years and vegans or strict vegetarians should consume foods fortified with vitamin B 12 or take vitamin B 12 supplements. Patients who have had bariatric surgery should receive 1 mg of oral vitamin B 12 per day indefinitely. Use of vitamin B 12 in patients with elevated serum homocysteine levels and cardiovascular disease does not reduce the risk of myocardial infarction or stroke, or alter cognitive decline.

Vitamin B 12 (cobalamin) is a water-soluble vitamin obtained through the ingestion of fish, meat, and dairy products, as well as fortified cereals and supplements. 1 , 2 It is coabsorbed with intrinsic factor, a product of the stomach's parietal cells, in the terminal ileum after being extracted by gastric acid 1 , 2 ( Figure 1 3 ) . Vitamin B 12 is crucial for neurologic function, red blood cell production, and DNA synthesis, and is a cofactor for three major reactions: the conversion of methylmalonic acid to succinyl coenzyme A; the conversion of homocysteine to methionine; and the conversion of 5-methyltetrahydrofolate to tetrahydrofolate. 1 , 2

In the United States and the United Kingdom, the prevalence of vitamin B 12 deficiency is approximately 6% in persons younger than 60 years, and nearly 20% in those older than 60 years. 1 Latin American countries have a clinical or subclinical deficiency rate of approximately 40%. 1 The prevalence is 70% in Kenyan school children, 80% in East Indian preschool-aged children, and 70% in East Indian adults. 1 Certain risk factors increase the prevalence of vitamin B 12 deficiency ( Table 1 ) . 4 , 5 Dietary insufficiency, pernicious anemia (i.e., an autoimmune process that reduces available intrinsic factor and subsequent absorption of vitamin B 12 1 , 2 , 6 , 7 ), and long-term use of metformin or acid-suppressing medications have been implicated in B 12 deficiency. 8 , 9

A multicenter randomized controlled trial of 390 patients with diabetes mellitus showed that those taking 850 mg of metformin three times per day had an increased risk of vitamin B 12 deficiency (number needed to harm = 14 per 4.3 years) and low vitamin B 12 levels (number needed to harm = 9 per 4.3 years) vs. placebo. 8 This effect increased with duration of therapy, and patients had an unclear prophylactic supplementation response. 8 A case-control study that compared 25,956 patients who had vitamin B 12 deficiency with 184,199 control patients found a significantly increased risk of vitamin B 12 deficiency in patients who had taken proton pump inhibitors (odds ratio = 1.65) or histamine H 2 blockers (odds ratio = 1.25) for at least two years. 9 In light of these findings, long-term use of these medications should be periodically reassessed, particularly in patients with other risk factors for vitamin B 12 deficiency. 8 , 9

Manifestations

Vitamin B 12 deficiency affects multiple systems, and sequelae vary in severity from mild fatigue to severe neurologic impairment 1 , 2 , 6 , 10 ( Table 2 4 , 10 ) . The substantial hepatic storage of vitamin B 12 can delay clinical manifestations for up to 10 years after the onset of deficiency. 11 Bone marrow suppression is common and potentially affects all cell lines, with megaloblastic anemia being most common. 1 , 2 , 6 The resultant abnormal erythropoiesis can trigger other notable abnormal laboratory findings, such as decreased haptoglobin levels, high lactate dehydrogenase levels, and elevated reticulocyte count. 1 , 2 , 6 Symptoms typically include being easily fatigued with exertion, palpitations, and skin pallor. 1 , 2 , 6 Skin hyperpigmentation, glossitis, and infertility have also been reported. 1 , 2 , 6 Neurologic manifestations are caused by progressive demyelination and can include peripheral neuropathy, areflexia, and the loss of proprioception and vibratory sense. Areflexia can be permanent if neuronal death occurs in the posterior and lateral spinal cord tracts. 1 , 2 , 6 , 12 Dementia-like disease, including episodes of psychosis, is possible with more severe and chronic deficiency. 1 , 12 Clinical evaluation seems to show an inverse relationship between the severity of megaloblastic anemia and the degree of neurologic impairment. 2

Maternal vitamin B 12 deficiency during pregnancy or while breastfeeding may lead to neural tube defects, developmental delay, failure to thrive, hypotonia, ataxia, and anemia. 4 , 13 – 16 Women at high risk or with known deficiency should supplement with vitamin B 12 during pregnancy or while breastfeeding. 4 , 14 – 16

Screening and Diagnosis

Screening persons at average risk of vitamin B 12 deficiency is not recommended. 17 Screening should be considered in patients with risk factors, and diagnostic testing should be considered in those with suspected clinical manifestations. 1 , 2 , 6 , 18

The recommended laboratory evaluation for patients with suspected vitamin B 12 deficiency includes a complete blood count and serum vitamin B 12 level. 2 , 19 – 21 A level of less than 150 pg per mL (111 pmol per L) is diagnostic for deficiency. 1 , 2 Serum vitamin B 12 levels may be artificially elevated in patients with alcoholism, liver disease, or cancer because of decreased hepatic clearance of transport proteins and resultant higher circulating levels of vitamin B 12 ; physicians should use caution when interpreting laboratory results in these patients. 22 , 23 In patients with a normal or low-normal serum vitamin B 12 level, complete blood count results demonstrating macrocytosis, or suspected clinical manifestations, a serum methylmalonic acid level is an appropriate next step 1 , 2 , 6 , 18 and is a more direct measure of vitamin B 12 's physiologic activity. 1 , 2 Although not clinically validated or available for widespread use, measurement of holotranscobalamin, the metabolically active form of vitamin B 12 , is an emerging method of detecting deficiency. 1 , 2 , 18 Table 3 lists the relative sensitivities and specificities of various laboratory tests. 24

Pernicious anemia refers to one of the hematologic manifestations of chronic auto-immune gastritis, in which the immune system targets the parietal cells of the stomach or intrinsic factor itself, leading to decreased absorption of vitamin B 12 . 1 Asymptomatic autoimmune gastritis likely precedes gastric atrophy by 10 to 20 years, followed by the onset of iron-deficiency anemia that occurs as early as 20 years before vitamin B 12 deficiency pernicious anemia. 25

Patients diagnosed with vitamin B 12 deficiency whose history and physical examination do not suggest an obvious dietary or malabsorptive etiology should be tested for pernicious anemia with anti-intrinsic factor antibodies (positive predictive value = 95%), particularly if other autoimmune disorders are present. 1 , 2 , 6 , 18 Patients with pernicious anemia may have hematologic findings consistent w ith normocytic anemia. 1 If anti-intrinsic factor results are negative but suspicion for pernicious anemia remains, an elevated serum gastrin level is consistent with the diagnosis. 2 The Schilling test, which was once the diagnostic standard for pernicious anemia, is no longer available in the United States. Figure 2 presents an approach to diagnosing vitamin B 12 deficiency and pernicious anemia. 18 , 26

Vitamin B 12 deficiency can be treated with intramuscular injections of cyanocobalamin or oral vitamin B 12 therapy. Approximately 10% of the standard injectable dose of 1 mg is absorbed, which allows for rapid replacement in patients with severe deficiency or severe neurologic sy mptoms. 2 Guidelines from the British Society for Haematology recommend injections three times per week for two weeks in patients without neurologic deficits. 18 If neurologic deficits are present, injections should be given every other day for up to three weeks or until no further improvement is noted. Table 4 lists the usual times until improvement for abnormalities associated with vitamin B 12 deficiency. 27 In general, patients with an irreversible cause should be treated indefinitely, whereas those with a reversible cause should be treated until the deficiency is corrected and symptoms resolve. 1 If vitamin B 12 deficiency coexists with folate deficiency, vitamin B 12 should be replaced first to prevent subacute combined degeneration of the spinal cord. 1 The British Society for Haematology does not recommend retesting vitamin B 12 levels after treatment has been initiated, and no guidelines address the optimal interval for screening high-risk patients. 18

A 2005 Cochrane review involving 108 patients with vitamin B 12 deficiency found that high-dose oral replacement (1 mg to 2 mg per day) was as effective as parenteral administration for correcting anemia and neurologic symptoms. 28 However, oral therapy does not improve serum methylmalonic acid levels as well as intramuscular therapy, although the clinical relevance is unclear. 29 There is also a lack of data on the long-term benefit of oral therapy when patients do not take daily doses. 2 There is insufficient data to recommend other formulations of vitamin B 12 replacement (e.g., nasal, sublingual, subcutaneous). 2 The British Society for Haematology recommends intramuscular vitamin B 12 for severe deficiency and malabsorption syndromes, whereas oral replacement may be considered for patients with asymptomatic, mild disease with no absorption or compliance concerns. 18

Because of potential interactions from prolonged medication use, physicians should consider screening patients for vitamin B 12 deficiency if they have been taking proton pump inhibitors or H 2 blockers for more than 12 months, or metformin for more than four months. 5 The average intake of vitamin B 12 in the United States is 3.4 mcg per day, and the recommended dietary allowance is 2.4 mcg per day for adult men and nonpregnant women, and 2.6 mcg per day for pregnant women. 30 Patients older than 50 years may not be able to adequately absorb dietary vitamin B 12 and should consume food fortified with vitamin B 12 . 30 Vegans and strict vegetarians should be counseled to consume fortified cereals or supplements to prevent deficiency. The American Society for Metabolic and Bariatric Surgery recommends that patients who have had bariatric surgery take 1 mg of oral vitamin B 12 per day indefinitely. 31

Vitamin B12 and Hyperhomocysteinemia

Vitamin B 12 deficiency is a much more common cause of hyperhomocysteinemia in developed countries than folate deficiency because of widespread fortification of food with folate. Although epidemiologic studies have shown an association between vascular disease and hyperhomocysteinemia, large randomized controlled trials have shown that lowering homocysteine levels in these patients does not reduce the number of myocardial infarctions or strokes, or improve mortality rates. 32 Similarly, an association between elevated homocysteine levels and cognitive impairment has been noted, but subsequent vitamin B 12 replacement does not have preventive or therapeutic benefit. 33

This article updates previous articles on this topic by Langan and Zawistoski , 4 and by Oh and Brown . 3

Data Sources : A PubMed search was completed in Clinical Queries using the key terms vitamin B 12 , cobalamin, deficiency, and treatment. The search included meta-analyses, randomized controlled trials, clinical trials, and reviews. Also searched were the Agency for Healthcare Research and Quality evidence reports, Clinical Evidence, the Cochrane database, Essential Evidence, the Institute for Clinical Systems Improvement, the National Guideline Clearinghouse database, and the U.S. Preventive Services Task Force. Search dates: March 1, 2016; October 20, 2016; and June 9, 2017.

Hunt A, Harrington D, Robinson S. Vitamin B 12 deficiency. BMJ. 2014;349:g5226.

Stabler SP. Clinical practice. Vitamin B 12 deficiency. N Engl J Med. 2013;368(2):149-160.

Brown DL, Oh R. Vitamin B 12 deficiency. Am Fam Physician. 2003;67(5):979-986.

Langan RC, Zawistoski KJ. Update on vitamin B 12 deficiency. Am Fam Physician. 2011;83(12):1425-1430.

Agency for Healthcare Research and Quality. Guideline summary: cobalamin (vitamin B 12 ) deficiency—investigation and management. January 1, 2012. https://www.guideline.gov/summaries/summary/38881 . Accessed October 13, 2016.

Dali-Youcef N, Andrès E. An update on cobalamin deficiency in adults. QJM. 2009;102(1):17-28.

Toh BH, van Driel IR, Gleeson PA. Pernicious anemia. N Engl J Med. 1997;337(20):1441-1448.

de Jager J, Kooy A, Lehert P, et al. Long term treatment with metformin in patients with type 2 diabetes and risk of vitamin B 12 deficiency: randomised placebo controlled trial. BMJ. 2010;340:c2181.

Lam JR, Schneider JL, Zhao W, Corley DA. Proton pump inhibitor and histamine 2 receptor antagonist use and vitamin B 12 deficiency. JAMA. 2013;310(22):2435-2442.

Derin S, Koseoglu S, Sahin C, Sahan M. Effect of vitamin B 12 deficiency on olfactory function. Int Forum Allergy Rhinol. 2016;6(10):1051-1055.

Carmel R. Current concepts in cobalamin deficiency. Annu Rev Med. 2000;51:357-375.

Reynolds E. Vitamin B 12 , folic acid, and the nervous system. Lancet Neurol. 2006;5(11):949-960.

Molloy AM, Kirke PN, Troendle JF, et al. Maternal vitamin B 12 status and risk of neural tube defects in a population with high neural tube defect prevalence and no folic acid fortification. Pediatrics. 2009;123(3):917-923.

Dror DK, Allen LH. Effect of vitamin B 12 deficiency on neurodevelopment in infants: current knowledge and possible mechanisms. Nutr Rev. 2008;66(5):250-255.

Centers for Disease Control and Prevention (CDC). Neurologic impairment in children associated with maternal dietary deficiency of cobalamin—Georgia, 2001. MMWR Morb Mortal Wkly Rep. 2003;52(4):61-64.

Hay G, Johnston C, Whitelaw A, Trygg K, Refsum H. Folate and cobalamin status in relation to breastfeeding and weaning in healthy infants. Am J Clin Nutr. 2008;88(1):105-114.

U.S. Preventive Services Task Force. A-Z topic guide. http://www.uspreventiveservicestaskforce.org/uspstopics.htm#AZ . Accessed May 24, 2016.

Devalia V, Hamilton MS, Molloy AM British Committee for Standards in Haematology. Guidelines for the diagnosis and treatment of cobalamin and folate disorders. Br J Haematol. 2014;166(4):496-513.

Carmel R, Green R, Rosenblatt DS, Watkins D. Update on cobalamin, folate, and homocysteine. Hematology Am Soc Hematol Educ Program. 2003:62-81.

Kaferle J, Strzoda CE. Evaluation of macrocytosis. Am Fam Physician. 2009;79(3):203-208.

Stabler SP, Allen RH. Megaloblastic anemias. In: Cecil RL, Goldman L, Ausiello DA, eds. Cecil Textbook of Medicine . 22nd ed. Philadelphia, Pa.: Saunders; 2004:1050–1057.

Arendt JF, Nexo E. Cobalamin related parameters and disease patterns in patients with increased serum cobalamin levels. PLoS One. 2012;7(9):e45979.

Andrès E, Serraj K, Zhu J, Vermorken AJ. The pathophysiology of elevated vitamin B 12 in clinical practice. QJM. 2013;106(6):505-515.

Oberley MJ, Yang DT. Laboratory testing for cobalamin deficiency in megaloblastic anemia. Am J Hematol. 2013;88(6):522-526.

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Pancytopenia due to vitamin b12 and folic acid deficiency—a case report.

1. Introduction

2. case presentation, 3. discussion, 3.1. pancytopenia and megaloblastic anemia in children, 3.2. general information on vitamin b12, 3.3. etiologies of vitamin b12 deficiency, 3.4. biological markers of vitamin b12 deficiency, 3.5. signs and symptoms of vitamin b12 deficiency, 3.6. therapeutic strategy, 3.7. folic acid deficiency, 3.8. vitamin deficiency among vegetarians and vegans, 4. conclusions, author contributions, institutional review board statement, informed consent statement, data availability statement, conflicts of interest.

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Depuis, Z.; Gatineau-Sailliant, S.; Ketelslegers, O.; Minon, J.-M.; Seghaye, M.-C.; Vasbien, M.; Dresse, M.-F. Pancytopenia Due to Vitamin B12 and Folic Acid Deficiency—A Case Report. Pediatr. Rep. 2022 , 14 , 106-114. https://doi.org/10.3390/pediatric14010016

Depuis Z, Gatineau-Sailliant S, Ketelslegers O, Minon J-M, Seghaye M-C, Vasbien M, Dresse M-F. Pancytopenia Due to Vitamin B12 and Folic Acid Deficiency—A Case Report. Pediatric Reports . 2022; 14(1):106-114. https://doi.org/10.3390/pediatric14010016

Depuis, Zoé, Sophie Gatineau-Sailliant, Olivier Ketelslegers, Jean-Marc Minon, Marie-Christine Seghaye, Myriam Vasbien, and Marie-Françoise Dresse. 2022. "Pancytopenia Due to Vitamin B12 and Folic Acid Deficiency—A Case Report" Pediatric Reports 14, no. 1: 106-114. https://doi.org/10.3390/pediatric14010016

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Megaloblastic anaemia in a 9-weeks old infant: A case report

Affiliation.

• 1 Department of Paediatrics, Federal Government Polyclinic (PGMI), Islamabad, Pakistan.
• PMID: 32400755
• DOI: 10.5455/JPMA.23379

Megaloblastic anaemia due to vitamin B12 and folic acid deficiency is uncommon in infancy and rarely reported in infants below 3 months of age. We hereby report a case of megaloblastic anaemia in a 9-weeks old infant having fever from 7th week of life. Blood picture showed pancytopenia and diagnosis was confirmed on bone marrow biopsy and serum level of vitamins. Patient positively responded to vitamin B12 and folic acid supplementation. Infants with pancytopenia even younger than 2 months, should also be investigated for vitamin B12 and folate deficiency. Mother of the baby was not antenatally investigated for anaemia. Prompt antenatal diagnosis and treatment of mothers can reduce the incidence in the infants.

Keywords: Megaloblastic anaemia, vitamin B12 deficiency, folic acid deficiency, infant.

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• Open access
• Published: 15 August 2014

Vitamin B 12 deficiency with combined hematological and neuropsychiatric derangements: a case report

• Luke Rannelli 1 ,
• Rita Watterson 2 ,
• Rupang Pandya 3 &
• Alexander A Leung 4  

Journal of Medical Case Reports volume  8 , Article number:  277 ( 2014 ) Cite this article

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Introduction

Although vitamin B 12 deficiency is a well-known cause of hematological and neuropsychiatric illness, the presentation of combined severe pancytopenia, demyelination and prominent psychiatric impairment is rare.

Case presentation

We present a case of a previously healthy 55-year-old East African man with severe vitamin B 12 deficiency (serum vitamin B 12 22pmol/L) secondary to pernicious anemia. He had a severe hypoproliferative megaloblastic anemia with hemolysis (hemoglobin 61g/L, mean corpuscular volume 99fL, reticulocytes 0.8%, haptoglobin undetectable), leukopenia (2.7×10 9 /L), thrombocytopenia (96×10 9 /L), ataxia with central demyelination, and megaloblastic madness. The patient’s anemia, myelopathy and psychiatric condition responded well to parenteral vitamin B 12 replacement therapy, with significant improvement seen within weeks.

Hematological manifestations of vitamin B 12 deficiency are typically inversely correlated with the presence and severity of neuropsychiatric impairment. Although uncommon, a presentation with severe hematological and neuropsychiatric disease can occur, as illustrated by this case. Its presence may help guide diagnosis as well as provide clinically important prognostic information.

Peer Review reports

Vitamin B 12 is essential for normal blood synthesis and neurological function. Vitamin B 12 deficiency may cause megaloblastic anemia, subacute combined degeneration of the cord and psychiatric illness (“megaloblastic madness”). Notably, the degree of bone marrow suppression is typically inversely related to both the presence and severity of neurological involvement. As such, the coexistence of significant anemia and neurological deficits is thought to be rare. We describe an unusual case of a patient with severe vitamin B 12 deficiency together with profound hematological derangements and florid neuropsychiatric impairment.

A 55-year-old man of East African descent presented to our community hospital with a history of repeated falls, postural dizziness, progressive fatigue, generalized weakness and 30-lb weight loss over the course of three to six months. He is a vegetarian. His collateral history obtained from family members confirmed a significant behavioral change over the past year, during which they observed irritability and emotional lability, as well as difficulty with self-care, grooming and personal hygiene. He was high-functioning before the onset of his present illness. During a review of systems, he denied any sensory deficits, visual impairment or taste perversion, and he also denied any history of bleeding, bruising or recent infection. His past medical history was unremarkable. He was not taking any medications at the time of presentation. The patient denied smoking, alcohol consumption or illicit drug use. He had no significant family history of any illness.

The initial examination in the emergency department revealed a poorly groomed, disheveled thin man who appeared older than his stated age. He was irritable, paranoid, delusional and highly circumstantial and had grandiose thoughts and impaired insight. He denied any hallucinations. The patient’s blood pressure was 96/50mmHg without postural change, his pulse was 92 beats/min and his respiratory rate was 16 breaths/min. He was afebrile. His neurological examination was limited because he was uncooperative, but he was observed to have an unstable, wide-based, ataxic gait as well as difficulty in performing rapid, alternating hand movements. His proprioception, vibration sense and light-touch sensory modalities appeared grossly preserved. His reflexes were symmetric and normal. His cardiovascular, respiratory, abdominal and dermatological examination results were unremarkable. Of note, he did not have evidence of splenomegaly, oral ulcers, nail changes or vitiligo. The patient was subsequently admitted to the hospital for further evaluation.

A complete blood count revealed hemoglobin 61g/L (mean corpuscular volume 99fL), thrombocytopenia 96×10 9 and leukopenia 2.7×10 9 . The patient’s reticulocytes were decreased at 14×10 9 /L (0.8%). A peripheral blood smear demonstrated hypersegmented neutrophils and schistocytes. His serum vitamin B 12 level was decreased at 22pmol/L (reference range 155pmol/L to 700pmol/L), his serum haptoglobin was undetectable and his serum lactate dehydrogenase was elevated at 1135U/L. The patient’s iron studies, folate, thyroid-stimulating hormone, liver enzymes, fibrinogen, serum protein electrophoresis, anti–tissue transglutaminase antibodies, hemoglobin A1c, coagulation panel, electrolytes and serum creatinine were all within normal limits. Viral studies for parvovirus B19, cytomegalovirus and human immunodeficiency virus were unremarkable. However, anti–intrinsic factor antibodies were detected. His blood type was B+. Of note, all blood work was performed prior to commencing any medical treatment or blood transfusions.

Computed tomography of the head revealed mild cerebral atrophy. Magnetic resonance imaging confirmed the presence of mild cerebral atrophy with increased signal intensity throughout the dorsal aspect of the cervical and thoracic spine, consistent with vitamin B 12 myelopathy (Figure  1 ). Chest radiography and computed tomography of the abdomen and pelvis were unremarkable. The patient did not consent to undergo diagnostic endoscopy. The patient was also unwilling to undergo electromyography with nerve conduction studies.

Magnetic resonance imaging scan of the patient’s spine. This sagittal view of the patient’s cervical and thoracic spine shows an area of hyperintensity (indicated by the arrow) along the dorsal column, consistent with demyelination due to vitamin B 12 deficiency.

The diagnosis of severe vitamin B 12 deficiency secondary to pernicious anemia was established on the basis of an unequivocally low serum vitamin B 12 level and the presence of anti–intrinsic factor antibodies. The patient’s long-standing vegetarian diet may also have been a contributor to his vitamin B 12 deficiency. Other causes of pancytopenia which may be characterized by a hypoproliferative, megaloblastic, macrocytic anemia—such as folate deficiency, hypothyroidism, viral illness and exposure to potential offending medications—were ruled out. The patient’s formal neurological and psychiatric evaluations were also consistent with central demyelinating disease and megaloblastic madness with prominent depressive features, after potential confounding metabolic and anatomical causes were excluded.

Because of the profound symptomatic anemia at presentation, the patient was initially given a transfusion of 2U of packed red blood cells upon admission. He was then initiated on daily subcutaneous injections of vitamin B 12 at 1000μg for one week, followed by weekly injections for the next four weeks, and he was subsequently prescribed monthly injections to be continued indefinitely. Within the first week of treatment, the patient’s pancytopenia had nearly resolved. His neuropsychiatric symptoms and cognition had also improved significantly by the time of hospital discharge. At follow-up three months later, the patient’s neuropsychiatric impairment had completely resolved. He denied any psychiatric disturbance, demonstrated a normal gait and had normal reflexes upon follow-up.

Vitamin B 12 deficiency is common, with an estimated prevalence of 15% in patients >60 years of age. Although most cases are clinically subtle, vitamin B 12 deficiency may sometimes present with florid disease [ 1 , 2 ]. When present, symptoms and signs can be broadly classified into hematological and neurological categories. Classically, vitamin B 12 deficiency may give rise to megaloblastic anemia and sub-acute combined degeneration of the spinal cord with demyelination of the dorsal column, resulting in both motor and sensory deficits. Less commonly, hemolysis and pancytopenia may be detected in the blood work, and neurological sequelae of optic nerve atrophy, autonomic nervous system dysfunction, peripheral neuropathy, as well as psychiatric complications of emotional lability, mania, paranoia, delusions, amnesia and psychosis, may also be present [ 3 ].

Notably, the degree of megaloblastic anemia in a patient with vitamin B 12 deficiency is inversely correlated with the presence and severity of neuropsychiatric symptoms at the time of presentation [ 3 – 6 ]. Severe anemia is rarely accompanied by any neurological symptoms or signs [ 5 ]. Likewise, hematocrit and mean corpuscular volume are commonly normal in patients with neuropsychiatric abnormalities [ 3 , 5 ]. The underlying reason for this relationship remains unclear. However, it is postulated that the mechanism responsible for the neurological dysfunction associated with vitamin B 12 deficiency is altogether different from those responsible for the hematological sequelae. Vitamin B 12 acts as a coenzyme for L-methylmalonyl-coenzyme A mutase and methionine synthetase. Accordingly, enzymatic defects resulting from vitamin B 12 deficiency lead to accumulation of methylmalonic acid and homocysteine, which appear to be proportionally related to the severity of the associated neurological and psychiatric abnormalities [ 4 ]. In contrast, inadequate vitamin B 12 results in ineffective DNA synthesis and impaired erythropoiesis, which account for the majority of the associated hematological derangements [ 3 ]. The underlying mechanisms responsible for the resulting megaloblastic anemia appear to be altogether different from the processes that produce the neurological sequelae of vitamin B 12 deficiency.

Myelopathy and peripheral neuropathy collectively account for the vast majority of neurological dysfunction [ 6 ], with mental impairment representing approximately 15% of cases [ 4 , 6 ]. Among patients with neurological disease, anemia and macrocytosis are the most common hematological derangements detected, and, when they are present, anemia and macrocytosis are typically minor and subtle [ 4 , 6 ]. In contrast, pancytopenia, severe anemia (that is, <60g/L), hemolysis, leukopenia and thrombocytopenia are all documented to be rare [ 2 , 3 , 5 ].

The presence of anemia also carries important prognostic significance. Anemia detected at baseline is inversely related to the severity of neurological impairment at diagnosis. Even after adjusting for differences in age, sex, mean corpuscular volume, serum vitamin B 12 level and disease duration, a lower hematocrit level remains an independent predictor of worse neurological disease [ 5 ]. It appears that the presence of anemia at baseline also favors a better neurological response to treatment and a more favorable long-term recovery [ 5 ].

Pernicious anemia can present with severe hematologic and neuropsychiatric conditions concurrently, contrary to stated dogma. Transient, self-limited neurological exacerbations may sometimes occur during the first few weeks of treatment. However, appropriate therapy can lead to complete recovery in up to two-thirds of patients treated [ 2 , 3 ].

Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.

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Acknowledgements

We would like to thank Dr Hasiba Imam and Dr Rosalyn McAuley, residents in internal medicine and psychiatry, who were involved quite closely with the patient’s care and documentation of his course in the hospital.

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Luke Rannelli

Department of Psychiatry, Foothills Medical Centre, University of Calgary, Special Services Building, 1403 29 Street NW, Calgary, AB, T2N 2T9, Canada

Rita Watterson

Department of Psychiatry, University of Calgary, 1926-3500 26 Avenue NE, Calgary, AB, T1Y 6J4, Canada

Rupang Pandya

Department of Medicine, University of Calgary, 313-4935 40 Avenue NW, Calgary, AB, T3A 2N1, Canada

Alexander A Leung

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LR, RW and AL were involved in drafting, writing and editing the manuscript. RP contributed to the writing and editing of the manuscript. All authors were directly involved in the care of the patient. All authors read and approved the final manuscript.

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Rannelli, L., Watterson, R., Pandya, R. et al. Vitamin B 12 deficiency with combined hematological and neuropsychiatric derangements: a case report. J Med Case Reports 8 , 277 (2014). https://doi.org/10.1186/1752-1947-8-277

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• Megaloblastic mania
• Neuropsychiatric
• Pancytopenia
• Pernicious anemia
• Vitamin B 12 deficiency

Journal of Medical Case Reports

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Article Contents

Case 2 diagnosis: megaloblastic anemia due to dietary cobalamin deficiency, clinical pearls, recommended reading.

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Case 2: A pale infant – not a typical case of iron deficiency

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Michelle P Wong, Louis Wadsworth, John K Wu, David Dix, Case 2: A pale infant – not a typical case of iron deficiency, Paediatrics & Child Health , Volume 13, Issue 6, July/August 2008, Pages 507–511, https://doi.org/10.1093/pch/13.6.507a

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A 15-month-old boy presented to his doctor when his mother became concerned about his diet. Despite the introduction of solids at six months of age, he was almost exclusively breastfed and a ‘picky eater’. His parents were partial vegetarians, with little meat intake. His mother had noticed that he was pale with gait disturbance and occasional falls. His developmental history was otherwise unremarkable. His past medical history included a term delivery with no complications and an uneventful postnatal period. After initial bloodwork, he was referred to a paediatric hematologist.

On examination, he was pale. His weight and height were between the 5th to 10th and 15th percentiles, respectively. A systolic murmur grade 2/6 was present. The patient's bloodwork included a complete blood count with white blood cell count 5.2×10 9 /L (normal 5.3×10 9 /L to 16×10 9 /L), neutrophils 1×10 9 /L (normal 1×10 9 /L to 6.5×10 9 /L), hemoglobin 83 g/L (normal 103 g/L to 135 g/L), mean corpuscular volume 107 fL (normal 70 fL to 86 fL) and platelet count 311×10 9 /L (normal 200×10 9 /L to 550×10 9 /L). His reticulocyte count was 31×10 9 /L (normal 40×10 9 /L to 120×10 9 /L), and his peripheral blood smear showed occasional oval macrocytes and hypersegmented neutrophils. A bone marrow aspirate was performed, and the images demonstrated classic findings of a particular diagnosis.

Additional investigations for macrocytic anemia included serum folate 33.3 nmol/L (normal greater than 6.8 nmol/L), and vitamin B 12 (cobalamin) 44 pmol/L (deficient range lower than 107 pmol/L). Based on the test results and the bone marrow aspirate findings of hypersegmented neutrophils, megaloblasts and giant metamyelocytes, a diagnosis of megaloblastic anemia (MA) was made.

Bloodwork was also performed on the mother. Serum folate was normal at 30.9 nmol/L (greater than 6.81 nmol/L) and serum vitamin B 12 was low-normal at 209 pmol/L (normal 133 pmol/L to 675 pmol/L). Maternal hemoglobin level and mean corpuscular volume (MCV) were normal.

Given the history, the likely cause of MA and cobalamin deficiency was reduced dietary intake. The toddler was treated with intramuscular cyanocobalamin injections (20 μg daily for seven days, then 100 μg weekly for four weeks) and a dietician was consulted. Oral cobalamin therapy was also an option and would have been effective, but for convenience and to ensure compliance, the parenteral route was chosen.

Two months later, he had improved significantly with a normal hemoglobin level (107 g/L) and MCV (83 fl). He was more energetic and interactive. His gait had returned to normal and his falls had decreased. His diet had improved with increased intake of meats, eggs, milk, fruits and vegetables, supplemented with PediaSure (Abbott Nutrition, Canada). Eight months after presentation, his development was appropriate for age and he was not anemic, despite being off therapy for several months. Maintenance of normal counts after discontinuation of therapy and adherence to a balanced diet are consistent with a dietary etiology for MA.

MA describes a group of disorders characterized by defective DNA synthesis. Morphological hallmarks in the marrow are megaloblasts and giant metamyelocytes displaying nuclear to cytoplasmic asynchrony. A megaloblast has a nucleus that is immature relative to the cytoplasm because the nucleus has impaired DNA synthesis, but hemoglobinization, which is dependant on ribosomal function, continues normally in the cytoplasm. There are many causes of MA, some of which are listed in Table 1 .

Causes of megaloblastic anemia

The case described demonstrates MA in a paediatric patient who was a ‘picky eater’ and was found to have macrocytic anemia. The insidious onset can delay diagnosis. It is important to diagnose this condition early to avoid the symptoms of anemia, as well as the neurological sequelae, including loss of vibration sensation and potential progression to spastic ataxia due to demyelination of the dorsal and lateral columns of the spinal cord. An approach to the workup of suspected MA in paediatric patients is proposed.

History and physical examination

Many patients are asymptomatic, and a diagnosis of MA is made incidentally when macrocytosis is found on routine bloodwork. There may be a history of poor food intake, prolonged breastfeeding, and a maternal history of vegetarian and vegan diets or autoimmune disorders. Nonspecific symptoms include irritability, weight loss, diarrhea or constipation. The clinical features are primarily those of a classic ‘lemon yellow’ pallor because of the combination of anemia and jaundice. Severe cases may have marked anorexia, weight loss, glossitis and angular cheilosis. Neurological effects may be manifested by failure to reach developmental milestones and may include paresthesias, muscle weakness and impaired intellectual development.

Screening bloodwork

The complete blood count shows macrocytic anemia. The MCV can be normal when there is concomitant microcytosis (thalassemia trait or iron deficiency). The peripheral blood smear shows oval macrocytes and hypersegmented neutrophils (five or more lobes) ( Figure 1 ). If a child is being breastfed, maternal bloodwork must be performed to exclude maternal vitamin deficiencies.

Peripheral blood smear demonstrating a hypersegmented neutrophil

Diagnostic tests

Folate and cobalamin levels are critical diagnostic blood tests. Red blood cell folate levels may be a better indicator of body folate because recent changes in dietary intake and hemolysis of the specimen will interfere with serum folate levels. Patients deficient in folate have low assay results, but a significant proportion of cobalamin-deficient patients will also have low red cell folate assays because cobalamin is a cofactor in folate metabolism. Cobalamin assays have limitations when correlating clinical deficiency with low-normal assay levels in some patients. Concentrations of the cobalamin carrier protein transcobalamin 1 can influence serum levels of vitamin B 12 . The assays for folate and vitamin B 12 levels are generally robust and convenient for diagnosing deficient states.

A bone marrow biopsy is an invasive procedure and less frequently performed with the availability of diagnostic blood tests, but it can be warranted to expedite diagnosis. The marrow can provide a morphological diagnosis within a matter of minutes; however, it requires skilled physician resources. A bone marrow aspirate in MA shows hypercel-lularity with ineffective hematopoiesis and megaloblastic erythropoiesis ( Figure 2 ), giant myeloid precursors (giant metamyelocytes) ( Figures 2 and 3 ), increased iron stores and, less commonly, hyperpolyploid megakaryocytes. When no explanation for cytopenias is found, bone marrow studies must be performed to exclude bone marrow failure, hematological malignancy or metastatic tumour.

Bone marrow aspirate demonstrating megaloblastic hematopoiesis with a megaloblast (white arrow) and a giant metamye-locyte (black arrow)

Bone marrow aspirate demonstrating megaloblastic hematopoiesis with a giant metamyelocyte (black arrow)

Additional tests and special tests

Schilling test:.

When Addisonian pernicious anemia (PA) is suspected, a Schilling test may be performed to assess cobalamin absorption. The test measures urinary excretion of orally administered radioactive cobalamin, with and without added intrinsic factor. PA is rare in children. The Schilling test would be helpful, and should be performed when there is a need to distinguish PA from the rarer malabsorptive errors of cobalamin absorption that are listed in Table 1 .

Total plasma homocysteine, serum methylmalonate and urinary excretion of methylmalonate:

Vitamin B 12 , but not folate, is required in methylmalonate (MMA) metabolism. Increased total plasma homocysteine and MMA levels are associated with cobalamin deficiency. Total plasma homocysteine level is elevated in both folate and vitamin B 12 deficiency, and is less specific than MMA. These tests, however, may be useful in suspected presymptomatic deficiency when the patient is not anemic and cobalamin levels are in the low-normal range. Elevated total plasma homocysteine and MMA levels may signal functional vitamin B 12 deficiency ( 1 ). Availability of these tests may limit their diagnostic application.

The etiology of folate or cobalamin deficiency must be determined because most causes are preventable or treatable. In developed countries, MA is more commonly caused by cobalamin rather than folate deficiency because many foods are folate-supplemented. Cobalamin deficiency must be ruled out before administering folic acid because treatment with folic acid alone, in the presence of cobalamin deficiency, can cause or exacerbate irreversible neurological damage.

MA can occur in children when there is a history of ‘picky eating’, poverty, chronic hemolysis such as hereditary spherocytosis, diets low in animal products or prolonged breastfeeding. Human milk cobalamin concentrations have been found to be lower in vegetarian mothers compared with omnivorous mothers ( 2 ). Breastfeeding mothers can have clinical ( 3 ) or subclinical cobalamin deficiency, the latter having low-normal cobalamin levels ( 1 ). As such, infants of cobalamin-deficient mothers are at risk of developing MA. Cobalamin deficiency may also occur in infants born to mothers with PA; however, this risk is largely theoretical because women with PA are usually infertile ( 4 ).

Early diagnosis of MA in childhood is imperative to prevent neurological consequences in infants who remain untreated. Increased awareness leading to early diagnosis and appropriate, timely therapy can prevent irreversible neurological effects for the child with MA. This is especially important during the critical period of neurodevelopment in early childhood.

Prolonged breastfed infants may be at risk of developing MA, especially if the mother consumes a vegetarian or vegan diet.

Cobalamin deficiency must be ruled out before instituting folate therapy.

Cobalamin deficiency may have neuropsychiatric as well as hematological manifestations, and early diagnosis is imperative to prevent irreversible neurological damage.

Measurement of serum MMA is important in the detection of presymptomatic cobalamin deficiency.

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• Published: 24 August 2024

Prevalence, risk factors, and treatment of anemia in hospitalized older patients across geriatric and nephrological settings in Italy

• Luca Soraci 1 ,
• Antonio de Vincentis 2 , 3 ,
• Filippo Aucella 4 ,
• Paolo Fabbietti 5 ,
• Andrea Corsonello 1 , 6 ,
• Elena Arena 2 ,
• Francesco Aucella 4 ,
• Giuseppe Gatta 4 &
• Raffaele Antonelli Incalzi 2 , 3  

Scientific Reports volume  14 , Article number:  19721 ( 2024 ) Cite this article

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Anemia is a common but often underdiagnosed and undertreated geriatric syndrome in hospitalized older patients. In this retrospective multicenter study, we aimed at characterizing the prevalence, risk factors, diagnostic and treatment approach to anemia in older patients admitted to acute care hospitals, focusing on differences between nephrology and geriatrics units. Prevalence and risk factors for anemia, diagnostic inertia (lack of iron, vitamin B12, and folate status assessment), replacement inertia (omitted treatment with iron, vitamin B12 or folic acid), and erythropoiesis-stimulating agents (ESA) inertia were explored. 1963 patients aged 82.7 (6.8) years were included in the study; 66.7% of the study population had anemia; among anemic patients, diagnostic inertia and replacement inertia were common with rates of 22–31% and 50–87%, respectively; omitted treatment with ESA affected 67.2% of patients and was more prevalent in geriatric units. In most cases, patients with ESA inertia were not routinely screened for iron tests. COPD, cancer, eGFR 45–60 ml/min were associated with increased tendency to ESA inertia. In conclusion, anemia had a high prevalence in older patients discharged from acute care units, but it is often underdiagnosed and undertreated.

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Introduction.

Anemia is one of the most common diseases in older individuals, as its prevalence increases with age 1 and is often underdiagnosed and undertreated, due to lack of specific symptoms in the older individual 2 . Aging-associated decline in serum hemoglobin concentration as well as increased prevalence of nutritional deficiencies and chronic diseases are the main pathophysiological factors contributing to the onset of anemia in older individuals 1 , 3 . The burden of anemia widely varies across distinct settings of care 4 , 5 , with mean prevalences ranging from 12% in community-dwelling individuals to up to 45% among hospitalized older patients 4 . Despite its relatively high prevalence, anemia cannot be considered a normal consequence of aging because it is associated with higher morbidity and mortality. Indeed, in the Cardiovascular Health Study, participants in the lowest hemoglobin quintile were characterized by increased mortality, even if not classified as anemic according to the WHO criteria 6 . Moreover, anemia has been associated with an array of clinical conditions such as depression, cognitive decline, decreased functional ability, increased risk of falls, longer hospital stays and decreased life expectancy 1 . Despite its clinical importance and impact on morbidity and mortality, diagnosis and treatment of anemia in the elderly is often suboptimal 7 .

Risk factors for anemia in older patients include age, comorbidities (i.e. diabetes mellitus, congestive heart failure [CHF], chronic obstructive pulmonary disease [COPD], and chronic kidney disease, CKD), inflammation, and malnutrition. Iron, folate, and/or vitamin B12 deficiencies are responsible for nearly one third of anemia cases in older patients; blood loss from gastrointestinal malignancies is the primary cause of iron deficiency in the elderly 8 ; however, routine blood tests for serum iron, ferritin and transferrin saturation have poor screening sensitivity for capturing iron deficiency in older patients 9 , 10 . Indeed, both age and concomitant inflammatory diseases tend to increase serum ferritin concentration above the reference level and may mask the presence of underlying anemic states 11 . Despite adaptation of transferrin saturation (TSAT) cut-offs has been proposed to increase sensitivity in diagnosing functional iron deficiency, it is not always implemented in routinary clinical practice 8 . Together with nutritional deficiencies and chronic inflammation, CKD is another major cause of anemia among older patients, accounting for up to one third of cases 8 . According to the KEEP study, the prevalence of CKD in people 65 years and older was approximately 44% and peaked in those over age 80; the great majority (77%) belonged to CKD stage 3 and only 5% to stage 4 12 ; recent analyses from the SCOPE study reported a CKD prevalence ranging from 39 to 66% depending on the equations used to estimate kidney function 13 .

Decrease in endogenous erythropoietin (EPO) production, absolute and/or functional iron deficiency, and inflammation with increased hepcidin levels 14 are the main mechanisms of anemia in CKD. For this reason, iron supplementation and erythropoiesis-stimulating agents (ESAs) are the mainstay of treatment in CKD-related anemia 15 . The remaining portion of older patients with are categorized as having unexplained anemia of aging (UAA), which is in part related to bone marrow abnormalities, including myelodysplastic syndrome 16 .

In any case, independent of underlying causes, appropriate assessment and management of anemia is often suboptimal 2 , and previous evidence hasraised this concern in community-dwelling cohorts 17 , 18 . Furthermore, in a study conducted by Rampotas et al. 19 in a cohort of over 800 patients with anemia admitted to acute care hospitals, among the 19% of patients who received inappropriate blood transfusions, over 50% did not have an aetiologic diagnosis of anemia before discharge. However, to date no study has investigated the quality of diagnostic and therapeutic approach to anemia in hospitalized older patients admitted to distinct medical specialties. Considering the increasing burden of multimorbidity in geriatrics units and of CKD-related anemia in nephrology units, the clinical and socioeconomic burden of anemia on hospitals is expected to increase, and both geriatricians and nephrologists are dealing with increasing rates of anemic patients.

Therefore, in this multicenter cross-sectional study we aimed at investigating the prevalence, risk factors, and quality of diagnostic and therapeutic approach to anemia in hospitalized older patients, focusing on differences between geriatric and nephrological practices.

Prevalence of anemia

Overall, the study population consisted of 1,903 patients aged 82.7 (7.8) years, with 51.9% men and a mean 7.3 (3.2) medications (Table 1 ).

Anemia had an overall prevalence of 66.7% of the study population and was slightly more common in Nephrology Units compared to Geriatric Units (75.6% vs 63.3%, p  <  0.001 ). Patients with anemia were older and more commonly men; furthermore, they had a higher prevalence of low iron tests, hypoalbuminemia and higher neutrophil-to-lymphocyte (NLR) values compared to those without anemia; they also had atrial fibrillation, type 2 diabetes, COPD, and cancer, as well as a lower mean eGFR value; finally, anemic patients took more medications, with higher prevalence of both antianemic and potentially anemizing ones. Characteristics of anemic patients stratified by admission unit are reported in Supplementary Table 1 . In summary, geriatric patients with anemia were older, with a higher prevalence of COPD, cardiovascular and cerebrovascular diseases, and a lower prevalence of type 2 diabetes mellitus compared to nephrology ones ( p  <  0.001 ). Moreover, patients admitted to Nephrology Units had a higher number of medications, a higher prescription of iron, vitamin B12, and folic acid. Prescription patterns slightly differed across admission units, partly in line with the median lower eGFR values in anemic patients discharged from nephrology units. Indeed, vitamin K antagonists and antiplatelet medications were more commonly prescribed in nephrology patients with anemia, while vitamin K antagonists had a higher prescription rate in geriatric units (Supplementary Table 1 ).

Characteristics of type and severity of anemia across admission units and CKD stages

Differences in severity and type of anemia in the overall study population and in the two settings are displayed in Fig.  1 .

Pie charts showing prevalence of anemia severities and types in the overall study population and across geriatrics and nephrology admission units.

Almost two thirds of anemic patients were characterized by a mild reduction of hemoglobin concentration with no significant differences across admission units; moderate anemia was slightly above 30% in all units, while severe anemia had a relatively low prevalence; among types of anemia, normocytic normochromic and mixed forms were the most common ones, followed by macrocytic and microcytic hypochromic forms. No significant differences emerged among patients admitted to Geriatric and Nephrology units.

Stratification by CKD stages showed that prevalence of anemia increases with declining eGFR in the overall study population, ranging from 53.4% to 57.0% to 71.5% to 83.9% to 83.5% for patients with eGFR ≥ 60, 45–59.9, 30–44.9, and < 30, respectively ( p  <  0.001 ). The distribution of severity and type of anemia across eGFR categories and admission units is represented in Fig.  2 .

Relative prevalence of anemia severities and types across eGFR categories in the overall study population and in the two admission units.

Decline of eGFR was associated with a graded increase in prevalence of moderate-severe anemia in overall study population and in both settings; more specifically, the most significant increase in the rate of moderate-severe anemia was observed for eGFR below 45 ml/min/1.73 m 2 in both settings; no significant changes of types of anemia across eGFR stages were observed.

Diagnostic inertia in all anemic patients and in those with indication to ESA treatment

Assessment of iron status, vitamin B12 and folic acid levels was performed infrequently in patients with anemia (Table 2 ).

Of all patients with anemia, only 30.6%, 23.4%, and 22.1% had iron studies, vitamin B12 and folic acid levels available, respectively. Patients admitted to Nephrology Units presented significantly higher rates of iron study availability (37.8 vs 27.8% %, p  <  0.001) and lower rates of vitamin B12 (16.3% vs 26.7%, p  <  0.001) , and folic acid assessment (15.6% vs 25.2%, p  <  0.001 ) . In general, among patients with anemia who underwent iron level assessment, more than a half resulted to have low iron tests, with higher prevalence in Nephrology Units compared to Geriatric Units (60.5% vs 55.3%, p  <  0.001 ), and an increasing trend in patients with eGFR < 30 ml/min/1.73 m 2 ; conversely, deficiencies of folic acid and vitamin B12 were less common.

Diagnostic inertia was also explored in patients with renal and hemoglobin criteria for ESA use (Table 3 ).

Overall, more than a half of patients satisfying these criteria were not routinely screened for iron status. Logistic regression analyses showing factors associated with lack of iron status assessment in bivariate and multivariate models are reported in Supplementary Table 2 . Factors associated with diagnostic inertia related to iron status were different across admission units (Supplementary Table 3 ); in this regard, among geriatric patients, CHF and prescription of DOACs resulted to be positively associated with diagnostic inertia, while number of drugs was negatively associated with the outcome; as expected, iron treatment was negatively associated with the outcome in overall study population and nephrology patients.

Replacement inertia

Replacement inertia was relatively common in the study population. Indeed, iron replacement inertia was frequently encountered among anemic patients with low iron levels, reaching the prevalence of 81.4% of the overall study population, 84.2% and 77.0% of patients hospitalized in geriatric and nephrology units, respectively ( p  =  0.244) . Among anemic patients with low serum folate, folate replacement inertia was found in 87.5% of all patients, ranging from 90.9% and 77.8% of geriatric and nephrology units respectively (p = 0.151). Finally, a half of the 12 patients with anemia and low vitamin B12 status were not treated with vitamin B12 supplements.

No factor was found to be associated with iron or replacement inertia in the overall study population (Supplementary Table 4 ).

• ESA inertia

Prevalence and risk factors for therapeutic ESA inertia among patients with ESA indication are reported in Supplementary Table 5 . ESA inertia was quite common in the study population (n = 219/326, 67.2%) with significantly higher rates in geriatric patients (173/203, 85.2% vs 46/123, 37.4%, p  <  0.001 ). In most cases, patients with ESA inertia were not routinely screened for iron tests (71.5%), especially in Geriatric units (75.4 vs 65.0%); in this subgroup of patients with ESA inertia and missed iron assessment, only a few were treated with intravenous/oral iron supplementation (10.8% in all units, 11.4% in geriatric units, 7.7% in nephrology units). Analyses of factors associated with ESA inertia in logistic regression bivariate and multivariate models are reported in Table 4 .

In the overall study population, COPD and increasing eGFR were associated with increased risk of ESA inertia in fully-adjusted models, while male sex, vitamin B12/folate treatment, and admission to Nephrology Units were negatively associated with the outcome in the study population. Stratified analyses by admission unit showed that eGFR and cancer were positively associated with ESA inertia among geriatric patients, while eGFR only was positively associated with the outcome in nephrology units.

To capture the complex non-linear relationship between eGFR, and ESA inertia, we fitted restricted cubic splines in the whole population, geriatric and nephrology units (Fig.  3 ).

Restricted cubic spline showing the association between eGFR (as measured through BIS equation) and the odds ratio of ESA inertia in patients with ESA indication (Hb < 10 g/dl, eGFR < 60 ml/min, and either normal-high iron status or low iron status with concomitant iron treatment), in the overall study population and in geriatrics and nephrology units separately. Odds ratios with 95% confidence intervals represent the risk of association compared to the median value of the eGFR distribution in the three study populations.

Overall, a trend of increased ESA inertia was present for less advanced CKD stages with specific cut-off of increased risk at eGFR > 37 ml/min/1.73 m 2 , significantly higher in nephrology vs geriatric patients (43 vs 40 ml/min/1.73 m 2 ). Logistic regression analyses using eGFR categorical variable > 45 ml/min/1.73 m 2 instead of continuous eGFR confirmed this association, with eGFR 45–60 ml/min/1.73 m 2 being the most consistent factor associated with ESA inertia across all settings in fully-adjusted models (OR 2.01, 1.51–3.01).

In this retrospective cohort study, we found a high prevalence of anemia and of diagnostic and therapeutic inertia with selected differences between Geriatrics and Nephrology Units. Prevalence of anemia was around 66.7% in the overall study population, with higher rates in nephrology units (75.6%) compared to geriatric units (63.3%); lower median eGFR values in nephrology units compared with geriatric units can partly explain the observed differences. Further differentiation showed that most anemias were mild in severity and more commonly of normochromic normocytic and mixed types. The present findings were consistent with those of previous studies reporting a prevalence ranging from 40–50% in community-dwelling cohorts 20 and up to 70–80% in older patients admitted to hospital 21 , 22 , 23 . As regards the severity and type of anemia, previous studies in out-of-hospital settings have already shown that mild anemias are considerably burdening older individuals, as they are often underdiagnosed and undertreated despite their relatively high frequency; indeed, more than one-tenth of individuals older than 65 are affected by anemia of mild degree, and this prevalence tends to exceed the 50% of the population older than 80 years, while more severe forms are less frequently detected 20 , 24 . Furthermore, while isolated iron-deficiency is recognized as the leading cause of anemia worldwide, and is common in older individuals due to malnutrition, decreased iron absorption, and gastrointestinal losses, microcytosis becomes poorly reliable in older patients, and ferritin is difficult to interpret since its levels tend to increase with age and inflammatory diseases 25 , 26 ; consequently, classical ferritin thresholds for younger patients need to be adapted in advanced age 1 ; in this regard, determination of serum level of soluble transferrin receptor (sTfR) and sTfR/ferritin index may help detecting iron deficiency in older patients with high ferritin values 3 , 27 , 28 as sTfR mediates the iron release to erythroid precursors and is less influenced by total inflammation 3 , 29 .

It is not surprising that older patients are more likely to experience normochromic normocytic anemias and mixed forms. Indeed, studies conducted in large cohorts of community-dwelling older patients from the National Health and Nutrition Examination Survey (NHANES) showed that anemia of chronic inflammatory diseases and unexplained forms represent over 50% of causes, while pure iron deficiency is detected in less than one-third of patients 20 , 24 ; similar data were found in studies conducted in hospitalized older patients, that were however monocentric or focused on CKD-related anemia only 21 , 22 , 30 .

Another relevant finding of the present study is that both diagnostic and therapeutic inertia were very commonly encountered in the hospital settings. Diagnostic inertia mainly refers to missed assessment of iron status (and, to a lesser extent, of vitamin B12 and folic acid levels) in patients with anemia; in this study, iron study, vitamin B12 and folic acid levels were checked only in approximately one-third of the study population, with slightly more frequent assessment in Nephrology Units; in patients with performed iron tests, an iron deficiency was detected in over 50% of cases, but it was infrequently corrected with appropriate iron supplementation. These results confirm previous findings in non-hospitalized populations 18 and in primary care patients with CKD 7 . In this regard, Xu et al. recently reported that iron status assessment is performed in less than 30% of patients with anemia and CKD stages 3–5 in the primary care setting 7 . Similarly, in a recent study on CKD patients and Hb < 10 g/dL, iron status was assessed with rates ranging from 25 to 47% 31 . This means that a substantial proportion of patients with anemia and CKD who could potentially benefit from iron supplementation are not being thoroughly examined and managed.

The prevalence and severity of anemia in this study showed an increasing trend with decreasing eGFR value, as observed in previous studies 32 , 33 ; this finding is pathogenically related to the effects of kidney function on erythropoiesis and hemoglobin production; CKD is a risk factor for anemia development and progression from mild-moderate to severe forms 30 , 34 . Impaired iron metabolism, decreased erythropoietin production, and increased hepcidin levels concur to the pathogenesis of anemia in CKD patients 14 . Diagnosis of anemia in CKD raises a significant burden on individual patients, due to its negative impact on quality of life 35 , 36 , increased morbidity and mortality 34 , 37 , 38 , as well as a rising economic burden on health care systems due to increased costs of care 39 and management complexity 31 . Indeed, the Chronic Kidney Disease Outcomes and Practice Patterns Study has shown that lower hemoglobin concentrations were associated with CKD progression and all-cause mortality 40 ; similarly, hemoglobin levels less than 11 g/dl were shown to impact health-related quality of life 41 , while a positive association with quality of life emerged for hemoglobin levels greater than 12 g/dl 40 .

The impact of anemia in geriatric populations raises the need for accurate diagnosis and appropriate management; in this regard, international KDIGO guidelines recommend to frequently check iron status levels, in order to choose the best therapeutic option for the individual patient, mainly represented by intravenous/oral iron and erythropoiesis stimulating agents (ESAs); indeed, the presence of low iron stores represents a clear indication to start iron treatment, which was previously shown to decreased need of ESA injection in CKD 42 . Conversely, in patients with hemoglobin < 10 g/dl and normal iron stores, start of ESA injection should be considered 42 .

Despite this evidence, however, both replacement and ESA inertia were highly common in the present study. In patients with low iron status, inertia to iron treatment was very common in our study, reaching up the 81.7% of the overall study population, with slightly higher rates in Geriatric vs Nephrology units, and remains high also after excluding patients taking ESAs (74%, 75%, and 71%, respectively); these findings consolidate previous evidence showing that only 10% of anemic patients in general are treated with oral/IV iron 7 ; folate and vitamin B12 inertia were also very common and characterized 87.5% and 50.0% of patients with low serum folate and vitamin B12 levels, respectively.

Furthermore, ESA inertia was also commonly encountered, with an overall prevalence of 67%, and important differences between geriatric (85%) and nephrology (37%) units; as already reported by Minutolo et al. 43 , inertia to ESA treatment was the second most common therapeutic inertia after iron replacement inertia; reluctance to treat anemia with ESA despite indication may be viewed in light of clinical trials demonstrating an increased risk of cardiovascular mortality and thrombotic events among patients with CKD treated with ESA 44 , 45 , that led the KDIGO consortium to consider the individualization of ESA treatment for patients under 10 g/dl of serum hemoglobin. It is not surprising that factors associated with ESA inertia included COPD and cancer, which may increase the production of red blood cells and the overall thrombotic risk, respectively. Inappropriately low ESA use was consistently observed for patients with eGFR between 45 and 60 ml/min, who generally require further assessment following discharge to define more clearly the exact kidney function, more independent of creatinine fluctuations which may occur in the hospital setting. Further research is needed in this field to adapt international guidelines to limit the risk of ESA underprescrition and misuse in older individuals, in order to decrease the prognostic impact of anemia in the geriatric population.

Strengths and limitations

This study has some strengths. First, it relies on a real-world setting population including older and multimorbid patients, with a high polypharmacy rate, that are generally excluded from prospective cohort studies or clinical trials. Additionally, this is the first study conducted in hospitalized older patients and showing a comparative analysis of the prevalence, risk factors, and diagnostic and therapeutic status of anemia in geriatric and nephrology acute care settings. On the other hand, some limitations need to be acknowledged. First, retrospective analyses were conducted on a single set of laboratory values rather than employing repeated measurements and clinical evaluations; consequently, it is not possible to ascertain whether abnormal laboratory parameters resulted from chronic or acute diseases. Second, we cannot exclude that reporting bias related to ICD coding systems has affected the differences in the distribution of diseases between patients with and without anemia; this might account for the unexpected low prevalence of COPD in patients with anemia, which contrasts with current literature. Similarly, the low prevalence of pulmonary embolism and deep vein thrombosis in the overall study population and, consequently, in patients with ESA inertia could be due to the same misreporting bias. Third, we cannot exclude that prevalence of iron, folate, and vitamin B12 assessment was underestimated due to lack of information on laboratory exams performed in the out-of-hospital setting, days or weeks before the hospitalization. Moreover, prevalence of iron deficiency may be underestimated, because of the high prevalence of comorbidities associated with increase in ferritin levels, such as CKD and cancer. However, use of age-adapted ferritin and TSAT thresholds could have blunted the risk of underestimation of iron deficiency in this population. Finally, a part of the increased rates of ESA inertia could be due to the fact that geriatricians might have sent patients to nephrology consultations in order to get ESAs prescribed.

Anemia significantly burdens older individuals discharged from geriatrics and nephrology acute care units, but it its pathogenetic mechanisms are explored in a minority of patients, and undertreatment is also highly prevalent, with selected differences between Geriatrics and Nephrology Units. Considering the detrimental effects of anemia on patients’ overall functioning and quality of life and on healthcare demands and expenses, efforts should be made to increase the attention and to improve the approach to anemia.

Data collection

The present study uses data from the Italian Society of Nephrology-Italian Society of Gerontology and Geriatrics (SIN-SIGG) study 46 , an observational, retrospective, cross-sectional study cooperatively funded by the Italian Society of Nephrology (SIN) and the Italian Society of Gerontology and Geriatrics (SIGG); this study aimed at collecting demographic, clinical and pharmacological information in patients aged 65 years or more consecutively admitted to twenty-four geriatric and fifteen nephrology units in Italy within the first half of 2018. The study design complies with the Declaration of Helsinki and Good Clinical Practice Guidelines. The study protocol was approved by the ethics committee of Italian National Institute for Health and Science on Aging (INRCA 18,018). All patients/participants provided their written informed consent to participate in this study.

All records and hospital discharges of patients aged 65 years or more admitted to the participating units have been reviewed without any other specific inclusion criteria. In case of multiple hospitalizations for the same patient during the identified observation period, only the first hospitalization has been included. Patients undergoing dialysis, those discharged within 24 h after hospital admission or who died during hospital stay were excluded from the study.

The sample size consisted of 2,202 patients aged 65 years or more to be initially included in the study. Patients with missing data for prescribed drugs (n = 111), serum creatinine (n = 34), and serum hemoglobin concentration (n = 154) were excluded, thus leaving a final sample of 1,903 patients. Characteristics of the study population were retrospectively retrieved by each medical record in participating institutions and included: demographic data (age, sex, body mass index), number and type of diseases and medications prescribed. All diagnoses were coded according to the International Classification of Diseases ninth Edition (ICD-9 Clinical Modification) system 47 and prescribed drugs at admission and discharge were assessed by the Anatomic Therapeutic Chemical (ATC) Classification System 48 . Polypharmacy was defined as taking 5 or more medications 49 .

Study variables

All patients underwent laboratory examinations during hospital stay. Anemia was defined as the presence of hemoglobin concentration < 12 g/dL in women and < 13 g/dL in men 50 . Anemia was further classified in relation to its severity (mild anemia: serum hemoglobin ≥ 10 g/dL; moderate anemia with hemoglobin < 10 and ≥ 8 g/dL; severe anemia with hemoglobin < 8 g/dL) and type of anemia according to mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC) (normocytic normochromic anemia: MCV equals to 80–95 fl and MCHC equals to 32–36 g/dL; microcytic hypochromic anemia: MCV < 80 fl and MCHC < 32 g/dL; macrocytic anemia: MCV > 95 fl; other types) 51 .

Laboratory parameters were also used to screen for distinct etiologies of anemia in our cohort; in particular, iron status was assessed by using serum ferritin and TSAT; low iron tests were defined as having ≤ 100 ng/mL or serum ferritin < 300 with serum transferrin saturation ≤ 20% according to age-adapted definition intended to increase diagnostic sensitivity in older people 52 , 53 . Vitamin B12 deficiency was defined as a serum vitamin B12 < 150 pg/mL 18 , while low folic acid was defined as having a folic acid ≤ 5 ng/mL 54 . Hypoalbuminemia, defined as having a albumin concentration less than 3.5 g/dL, was assessed a s a surrogate marker of malnutrition 55 . Serum creatinine levels were used to assess renal function. The estimated glomerular filtration rate (eGFR) was calculated by using Berlin-Initiative-Study 1 (BIS1) equation 56 :

where S Cr stands for serum creatinine. CKD stages were categorized as follows: eGFR ≥ 60, 45–59.9, 30–44.9, < 30 ml/min/1.73 m 2 .

We verified whether iron status, vitamin B12, and folic acid were assessed in anemic patients of both admission units, in order to capture the risk of diagnostic inertia. Therapeutic inertia was systematically assessed and defined as omitting to correct iron/B12/folate deficit (replacement inertia) or supplementing ESA when indicated (ESA inertia); indication to ESA prescription was defined as having hemoglobin < 10 g/dl, eGFR < 60 ml/min/1.73 m 2 and either normal iron status or low iron status and presence of iron supplementation 57 , 58 .

Statistical analysis

Descriptive analysis was first conducted to compare patients with and without anemia regarding demographic, clinical, pharmacological, and laboratory characteristics. Continuous variables were reported as either mean and standard deviation (SD) or median and interquartile range (IQR), according to their distribution evaluated by visual inspection and Kolmogorov–Smirnov test; categorical variables were reported as number and percentage (%). The two-paired sample t-test and Mann–Whitney U test were used to compare continuous categorical variables, while the two-tailed Chi-squared Pearson’s test was used to compare categorical variables. Subgroup descriptive analyses were conducted in geriatrics and nephrology units separately, to account for differences in the prevalence and risk factors for anemia across admission units.

We investigated potential predictors of replacement and ESA inertia, based on previous knowledge from the literature and clinical relevance: demographic characteristics, comorbidities known to be associated with cardiovascular risk and thrombosis (hypertension, atrial fibrillation, CHF, coronary artery disease [CAD], cerebrovascular disease [CVD], diabetes mellitus, chronic obstructive pulmonary disease [COPD], diabetes mellitus, cancer), eGFR and the use of concomitant medications potentially causing anemia (antiplatelets, anticoagulants, PPI, corticosteroids) 57 , 58 , 59 . Assessment of risk factors associated with ESA inertia was also explored by fitting logistic regression models including covariates retrieved by literature review. Collinearity among covariates included in the multivariate models was verified by the Spearman’s rank correlation coefficient and the variance inflation factor (VIF). A  p-value  < 0.05 was considered statistically significant. To explore the non-linear relationships between ESA inertia and continuous predictors, such as eGFR, albumin, and NLR, we fitted restricted cubic splines with number of knots selected through Akaike’s information criteria (AIC) and according to the best model fit evaluated with Wald Chi-squared test. Statistical analysis was performed using the R version 4.6.

Data availability

The raw data supporting the conclusion of this article will be made available by the authors without undue reservation (contact name Luca Soraci, Unit of Geriatric Medicine, IRCCS INRCA, [email protected]).

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Acknowledgements

The authors thank the Italian Society of Gerontology and Geriatrics (SIGG) and the Italian Society of Nephrology (SIN) collaborators of the participating geriatrics and nephrology units (SIN-SIGG group, supplementary information ).

This work was partially supported by the Ricerca Corrente funding from the Italian Ministry of Health to IRCCS INRCA.

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Soraci, L., de Vincentis, A., Aucella, F. et al. Prevalence, risk factors, and treatment of anemia in hospitalized older patients across geriatric and nephrological settings in Italy. Sci Rep 14 , 19721 (2024). https://doi.org/10.1038/s41598-024-70644-8

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Long-Term Zidovudine Therapy and Whether It is a Trigger of Vitamin B12 Deficiency: A Case Study of Megaloblastic Anemia at the University of Zambia Teaching Hospital

Natasha mupeta kaweme.

University Teaching Hospital, Private Bag RW1X Ridgeway, Nationalist Road, Lusaka 10101, Zambia

Sahar Mounir Nagib Butress

Hamakwa muluti mantina, associated data.

All data underlying the results are available in the article, and no additional source data are required.

Macrocytic anemia is frequently observed in adult HIV-infected patients treated with nucleoside reverse transcriptase inhibitors and with vitamin B12 and folate deficiency. In this case report, we discuss a 52-year-old nonvegetarian male on long-term antiretroviral therapy for 5 years, presenting with severe macrocytic anemia (hemoglobin, 3.7 g/dL; mean corpuscular volume, 119.6 fL) and leukopenia (2.71 ∗ 10 9 /L), who was diagnosed with megaloblastic anemia caused by vitamin B12 deficiency following laboratory investigations. Parenteral vitamin B12 replacement therapy was initiated, with an early response observed. Notwithstanding, the treatment response was not sustained as the patient later presented with refractory anemia and persistence of macrocytosis. Discontinuation of zidovudine with concurrent vitamin B12 administration promptly improved the patient's clinical deficiency symptoms. At the end of 3 months, the patient had a complete hematological recovery. The deficiency of vitamin B12 disrupts DNA synthesis inhibiting effective hematopoiesis in all cell lines, particularly erythroid precursors and further promotes reversible bone marrow failure. Long-term ART therapy with zidovudine causes cytotoxicity in myeloid and erythroid precursors and induces bone marrow suppression. Whether long-term zidovudine consumption induced lower levels of vitamin B12 and subsequent megaloblastic anemia requires in-depth research and exploration.

1. Introduction

Anemia is a frequent hematological complication of human immunodeficiency virus (HIV) infection, and its incidence is adversely associated with advanced HIV disease, poor treatment outcomes, and increased risk of patient mortality [ 1 , 2 ]. Although the causes of anemia in the general population are multifactorial in origin with various pathophysiological mechanisms, the most common etiology globally is iron deficiency, among other nutritional deficiencies such as folic acid and vitamin B12 [ 1 ]. In the setting of HIV infection, several factors, including chronic disease, the direct/indirect influence of HIV infection on hematopoietic stem/progenitor cells [ 3 ], altered cytokine milieu, opportunistic infections, and ART (antiretroviral therapy)-induced toxicity, result in impaired hematopoiesis and suppressed proliferation and differentiation of hematopoietic stem/progenitor cells, thus leading to cytopenias, anemia, thrombocytopenia, and neutropenia [ 4 – 6 ].

Although the burden of anemia among persons living with HIV in Zambia has not been fully established, a recent study by Cao et al. reported that the prevalence of anemia among adults living with HIV was higher in Southern Africa than that in East Africa. Furthermore, anemia in HIV infection was common among all groups in the population [ 2 ] and was higher in advanced HIV disease, among women, infants, and children in developing countries [ 7 , 8 ]. Other factors, including socioeconomic factors, marital status, monthly income, and educational status, influence the severity of anemia among persons living with HIV [ 9 ].

Macrocytic anemia, defined as an increase in red blood cell mean corpuscular volume (MCV) of greater than 100 femtoliters (fL) in the setting of anemia, is a key observation in patients on long-term nucleoside reverse transcriptase inhibitors (NRTIs) [ 10 , 11 ]. The exact mechanism by which NRTIs such as zidovudine led to macrocytosis in HIV-infected persons is yet to be explored. NRTIs interfere with mitochondrial DNA synthesis by inhibiting polymerases responsible for erythrocyte formation. Interference with mitochondrial DNA synthesis impairs the synthesis of erythroid precursors in the bone marrow (megaloblastic erythropoiesis) and consequently, macrocytosis [ 12 – 14 ]. Similarly, Reddy et al. hypothesized that zidovudine suppressed erythropoiesis and inhibited erythroid stem cells resulting in red cell aplasia, decreased reticulocyte counts and hemoglobin levels without hemolysis or blood loss [ 15 ], and increased MCV and erythropoietin levels [ 16 ]. A study conducted 3 decades ago highlighted that life-threatening hematological toxicity was probably attributed to transient depletion of mitochondrial DNA and sensitivity of DNA polymerase in some cell mitochondria [ 17 ].

At present, zidovudine and stavudine are less commonly used, as novel ART combinations with safer toxicity profiles have been introduced. In pediatric patients, zidovudine has been extensively used for treating HIV and preventing perinatal transmission of HIV in neonates [ 18 ]. However, the adverse mitochondrial toxicity of zidovudine favors the use of abacavir or tenofovir alafenamide. Zidovudine-induced bone marrow toxicity is common in patients with advanced disease and is related to early long-term high-dose therapy [ 19 ]. Generally, stavudine is associated with significant toxicity and increases the risk of macrocytosis, limiting its use in clinical practice [ 10 ].

According to the Zambia Consolidated HIV guidelines 2020 (currently in use), zidovudine-based regimens are the preferred second-line regimen for patients failing on tenofovir or abacavir-based regimens. A major concern for this arises from a recent study in Zambia that demonstrated high prevalence of HIV drug resistance, including thymidine analog mutations that confer resistance or reduced susceptibility to zidovudine, stavudine, and other NRTIs in adolescents and young adult patients [ 20 ].

This case study will discuss our experience with a patient who presented with macrocytic anemia associated with a history of long-term zidovudine consumption coupled with vitamin B12 deficiency and hypothesize the importance of a pragmatic approach to the diagnosis and management of macrocytic anemia in persons living with HIV to ensure the provision of quality health care in a resource-limited setting.

2. Case Presentation

A 52-year-old nonvegetarian male was referred to our hematology department with a one-month history of swelling of both lower limbs, progressive generalized body weakness, and severe anemia. He was diagnosed with HIV infection in 2010 and was on antiretroviral drugs for approximately 11 years at the time of presentation. The patient reported that his initial regimen was tenofovir/emtricitabine/efavirenz. He had no prior history of alcohol consumption and reported no HIV-related opportunistic infections during his course of treatment. His physical examination was significant for severe palmar and conjunctival pallor.

Initial laboratory results showed bicytopenia, hemoglobin 3.7 g/dL (reference range, 14.3–18.3), white cell count 2.71 ∗ 10 9 /L (reference range, 4.00–10.00), and a normal platelet count of 301 ∗ 10 9 /L (reference range, 150–400) with an elevated mean corpuscular volume (MCV) and mean cell hemoglobin (MCH) of 119.6 femtoliters (fL) (reference range, 79.1–98.9) and 40.2 picograms (pg) (reference range, 27.0–32.0), respectively. Peripheral blood differential counts were 55.3% neutrophils, 33.6% lymphocytes, 10.7% monocytes, 0.4% eosinophils, and no basophils. The peripheral blood smear showed anisopoikilocytosis, ovalomacrocytosis, and hypersegmented neutrophils. Serum vitamin B12 was 66.2 pg/ml (reference range, 211–946), and serum folate was 7.75 ng/ml (reference range 4.6–34).

A comprehensive metabolic panel, including kidney function tests, liver function tests, and a lipid panel, was within the normal range. Additional investigations such as stool for occult blood and echocardiogram/electrocardiogram showed no abnormalities. The patient had a complete viral suppression (target not detected) with an absolute CD4 count of 451 cells/ µ L (reference range, 410–1590).The laboratory test results indicating a significant deficiency of vitamin B12 (66.2 pg/ml) and peripheral blood smear findings of hypersegmented neutrophils and oval macrocytes led the team to the diagnosis of megaloblastic anemia.

The patient received 4 units of packed cells while admitted, and parenteral vitamin B12 at 1000 mcg daily was initiated. At subsequent clinic visits, recovery of blood cell counts with normalization of MCV and MCH parameters was noted. The serum intrinsic factor antibody test result was equivocal. Despite this, maintenance of vitamin B12 at weekly and then monthly dosing was continued.

Two months after initiation of vitamin B12, the patient presented to our outpatient clinic with a complaint of generalized body weakness and heart palpitations with severe conjunctival pallor and tachycardia on examination. A complete blood count showed severe anemia with hemoglobin 3.6 g/dl, white cell count 2.41 ∗ 10 9 /l, MCV 114.1 fL, MCH 36.4 pg, and a normal platelet count. Peripheral blood smear findings were consistent with the initial report but showed significant numbers of round macrocytes, occasional oval macrocytes, and hypersegmented neutrophils. The patient's laboratory workup is summarized in Table 1 .

Laboratory results at initial diagnosis and at follow-up visits.

After a detailed review of the patient's drug history, it was found that his antiretroviral medication was changed to an AZT-based regimen (zidovudine/lamivudine/efavirenz) in 2017 for unknown reasons. We decided to admit the patient for packed cell transfusion, and a consult was sent to the infectious disease unit to revise the treatment regimen. Following transfusion, the patient was discharged on vitamin B12, folic acid, and a dolutegravir-based regimen (tenofovir disoproxil fumarate/lamivudine/dolutegravir) based on the current WHO recommendations for first-line and second-line antiretroviral regimens. The patient was closely followed up in the outpatient department and experienced complete recovery of hemoglobin, red cell count, MCV, and MCH count with an improving absolute neutrophil count within 3 months. At present, the patient reports good physical health with stable blood counts.

3. Discussion

Macrocytic anemia is broadly classified as [ 1 ] megaloblastic associated with vitamin B12 and folate deficiency and [ 2 ] the nonmegaloblastic moiety associated with chronic alcoholism, liver disease, hypothyroidism, hereditary spherocytosis, and states of increased red cell consumption such as hemolysis or high turnover in pregnancy [ 21 ]. The most common cause of megaloblastic anemia is vitamin B12 deficiency caused by malabsorption due to the absence of intrinsic factors caused by pernicious anemia or following gastric surgery, transcobalamin II deficiency, duodenal and colonic inflammatory damage from celiac disease, or infection with the tapeworm Diphyllobothrium latum , and insufficient dietary intake. Common drugs such as phenytoin, trimethoprim, valproic acid, hydroxyurea, methotrexate, azathioprine, zidovudine, and other antiretroviral drugs can also cause macrocytic anemia [ 22 ].

Early identification and prompt treatment of vitamin B12 deficiency, a reversible and treatable cause of ineffective hematopoiesis, can significantly improve the survival outcomes of persons living with HIV. Our patient experienced refractory anemia and mild leukopenia despite continued treatment with parenteral vitamin B12 with no concern for other nutritional factors, opportunistic infections, or progression of HIV disease. At the initial diagnosis and confirmation of vitamin B12 deficiency and megaloblastic anemia, the patient showed an early response to vitamin B12 supplements; however, this early response was not sustained. We hypothesized that the relapse of megaloblastic anemia was probably associated with significantly low vitamin B12 levels, higher MCV levels, and the persistence of mitochondrial toxicity or the presence of mitochondrial DNA mutations induced by zidovudine which might elicit more severe mitochondrial dysfunction [ 21 ].

Although the patient's folate level of 7.75 ng/ml (reference range 4.6–34) was within the normal range (lower limit of normal), concern for ‘masked' folate deficiency after initiation of vitamin B12 was speculated. Vitamin B12 deficient cells are unable to utilize folate, and only upon treatment with vitamin B12, folate can be utilized. As a result, underlying folate deficiency can manifest with a significant decrease in folate levels of less than 2 ng/ml. Concurrent administration of folate with vitamin B12 will avert a suboptimal response of anemia to vitamin B12 alone. Measurement of red cell folate assay, serum methylmalonic acid, and homocysteine levels could have confirmed the likelihood of folate deficiency or combined nutritional deficiency. However, the tests were not performed due to financial constraints. In vitamin B12 deficiency, both metabolites increase proportionally to the severity of deficiency, whereas in pure folate deficiency, only homocysteine levels are elevated and methylmalonic acid is normal [ 23 ].

Desk review has not revealed any study that has explored the possibility of an association between zidovudine therapy and lower levels of vitamin B12 and folate and its impact on hematopoiesis and erythropoiesis. Notwithstanding, Puspasari et al. hypothesized that interference of mitochondrial DNA synthesis induced by zidovudine resulted in hematological interference in the form of reduced vitamin B12 and folate levels in blood which in turn, led to megaloblastic anemia and oral lesions [ 24 , 25 ].

Cotrimoxazole prophylaxis is commonly used in Zambia for the prevention of pneumocystis jirovecii pneumonia, isosporiasis, toxoplasmosis, malaria, and other HIV-related and non-HIV-related diseases. It is initiated in adult patients with a CD4 count of less than 350 cells/ µ L or having stage II, III, or IV disease according to the WHO clinical staging and discontinued when the CD4 count is greater than or equal to 350 cells/ µ L for two consecutive values at least 6 months apart while on ART (Zambia Consolidated Guidelines for Treatment and Prevention of HIV Infection, 2020). Trimethoprim is considered a weak inhibitor of dihydrofolate reductase that can interfere with folic acid metabolism and promote megaloblastic changes at high doses [ 7 ]. In this case study, the patient reported no history of cotrimoxazole consumption; therefore, it was excluded as a possible cause of megaloblastic anemia.

Neutropenia is a common concomitant feature in megaloblastic anemia. Vitamin B12 and folate deficiency interferes with effective hematopoiesis and induces bone marrow suppression [ 26 ], decreased myeloid production, and adverse neutropenia which increases the risk of infection. Other etiologies, including bacterial and viral infections, immunodeficiency and underlying chronic disease, are commonly associated with leukopenia and neutropenia. In patients with infection due to neutropenia, underlying vitamin B12 deficiency may exacerbate the susceptibility to cytopenias [ 27 ].

Our patient had chronic but asymptomatic neutropenia, persisting for over three months. Notwithstanding, a gradual improvement in the total white cell count was observed with vitamin B12 treatment and zidovudine discontinuation. We hypothesized that the presence of chronic neutropenia could have been a result of prolonged or advanced vitamin deficiency in underlying HIV infection, although the patient had complete viral suppression. About 4 decades ago, Kaplan and Basford hypothesized that megaloblastic anemia of vitamin B12 and folate deficiency was associated with morphological and quantitative abnormalities in developing leucocytes, resulting in reduced leucocyte numbers with nuclei hypersegmentation [ 28 ].

Appropriate treatment of vitamin B12 deficiency provides rapid resolution of clinical deficiency symptoms and prompt hematological recovery. Our patient's biological markers reached normal values or improved within 3 months of zidovudine discontinuation and vitamin B12 supplementation. Furthermore, the patient had no clinical features consistent with deficiency. Initial response to vitamin B12 can be determined by reticulocytosis occurring within a week after starting treatment. Complete hematological recovery and normalization of MCV are estimated to be between 2 and 8 weeks [ 29 ]. The persistence of macrocytosis is not affected by the duration of zidovudine therapy. Yu et al. showed that a higher MCV took longer to normalize, with a percentage of patients having persistent macrocytosis 2 years after zidovudine cessation [ 21 ].

The treatment and follow-up of patients on antiretroviral drugs require a multidisciplinary approach, including ART specialists, hematologists, nutritionists, and pharmacologists, to ensure early identification of drug-induced macrocytosis and prompt intervention. Early nutritional intervention can ensure optimal nutrition and health status in persons receiving ART. Rezaei et al. suggested that combined administration of vitamin B12 and folate supplements had a beneficial effect on the hematological status of HIV-infected persons receiving HAART [ 30 ]. An urgent revision of first-line and second-line therapies to include novel integrase inhibitors such as dolutegravir, also currently available in resource-limited settings, can reduce the incidence of zidovudine-related macrocytic anemia. Zidovudine is avoided in patients with low hemoglobin levels due to its additive effect on anemia and myelotoxicity. Monthly monitoring of hemoglobin levels after switching and re-initiating ART drugs for three months should be mandatory.

Among the significant limitations in the management of our patient were the inability to measure serum methylmalonic acid and homocysteine levels to increase the sensitivity and precision of the diagnosis of vitamin B12, folate, or combined deficiency. These metabolites are useful markers when the clinical picture is equivocal. Second, a reticulocyte count was not possible, given the unavailability of methylene blue stain. Reticulocytopenia suggests vitamin B12 deficiency in megaloblastic anemia. The seminal limitation was the inability to assess vitamin B12 and folate serum levels following treatment to monitor response. The recent literature has revealed that monitoring vitamin B12 is not necessary for patients receiving replacement therapy.

4. Conclusion

Macrocytic anemia is frequently observed in HIV patients on long-term nucleoside reverse transcriptase inhibitors such as zidovudine. In this case study, we have discussed the history, presentation, and laboratory workup of a patient with refractory anemia and elevated mean corpuscular volume and mean corpuscular hemoglobin on long-term zidovudine therapy coupled with vitamin B12 deficiency, thus supporting a diagnosis of megaloblastic anemia. It may be possible that increased zidovudine toxicity was exacerbated by vitamin B12 deficiency, as each is associated with bone marrow suppression. Discontinuation of zidovudine and treatment with parenteral vitamin B12 resulted in a rapid resolution of the patient's clinical deficiency symptoms.

It is evident that anemia is prevalent among people living with HIV infection, and therefore, more attention is required for a tailored, safer choice of ART regimens to minimize drug-related hematological toxicities. It is highly recommended that older treatment regimens be discarded in preference for safer novel regimens that have demonstrated improved survival outcomes while minimizing adverse events. Accordingly, we propose a detailed study that will explore the link between long-term zidovudine therapy and lower levels of vitamin B12 and folate.

Acknowledgments

The authors thank the entire hematology department for their continued support and Mr. Elemson Muyanga for his dedicated assistance in editing the final manuscript.

Abbreviations

Data Availability

Conflicts of interest.

The authors declare that they have no conflicts of interest.

Authors' Contributions

NMK performed literature research and wrote the final manuscript. SMNB devised the study and conducted a critical review of the manuscript. HMM conceived and revised the final manuscript. All authors read and approved the final manuscript.

Metastatic Adenocarcinoma to Bone Marrow Presenting as Microangiopathic Hemolytic Anemia – Diagnostic Issues in a Series of Seven Young Adults

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• Published: 24 August 2024

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• Babu Vishva   ORCID: orcid.org/0009-0008-3098-8458 1 ,
• Arumugam Pradeep   ORCID: orcid.org/0000-0002-7470-1833 1 ,
• Nimesh Mishra   ORCID: orcid.org/0009-0004-9496-398X 1 ,
• Rakhee Kar   ORCID: orcid.org/0000-0001-6041-1512 1 &
• Debdatta Basu   ORCID: orcid.org/0000-0001-5096-1406 1   nAff2  

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Cancer related microangiopathic hemolytic anemia is a rare entity and shares clinical and hematological features with other causes of thrombotic microangiopathy and leads to diagnostic dilemma and treatment delays. This record-based descriptive study, spanning five years, details the clinicopathological features with special emphasis on the peripheral blood findings in unsuspected cases of bone marrow metastasis of unknown primary in young adults. Seven relatively young patients, with an average age of 27 years, presented with unexplained anemia with peripheral blood showing features of microangiopathic hemolytic anemia, thrombocytopenia, and leukoerythroblastic picture. Subsequent bone marrow examination revealed presence of metastatic adenocarcinoma with the primary site being detected in five of the seven patients. This case series highlights these uncommon, but significant, hematological manifestations of metastatic adenocarcinoma in bone marrow in young adults, and the importance of astute observations of peripheral blood smear in detection of an underlying malignancy.

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Debdatta Basu

Present address: Mahatma Gandhi Medical College and Research Institute, Pondicherry, 607402, India

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Department of Pathology, JIPMER, Pondicherry, 605006, India

Babu Vishva, Arumugam Pradeep, Nimesh Mishra, Rakhee Kar & Debdatta Basu

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Vishva, B., Pradeep, A., Mishra, N. et al. Metastatic Adenocarcinoma to Bone Marrow Presenting as Microangiopathic Hemolytic Anemia – Diagnostic Issues in a Series of Seven Young Adults. Indian J Hematol Blood Transfus (2024). https://doi.org/10.1007/s12288-024-01842-7

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Received : 17 June 2024

Accepted : 04 August 2024

Published : 24 August 2024

DOI : https://doi.org/10.1007/s12288-024-01842-7

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