What is the appropriate diagnostic workup for anemia?

How to Diagnose Anemia

Define Anemia Using WHO Criteria

Begin by confirming anemia with hemoglobin thresholds: <13 g/dL in men, <12 g/dL in non-pregnant women, and <11 g/dL in pregnant women. 1

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Essential Initial Laboratory Panel

Order a complete blood count with red cell indices (MCV, MCH, MCHC, RDW), absolute reticulocyte count, serum ferritin, transferrin saturation, and C-reactive protein as your minimum diagnostic workup. 1, 2

This combination allows you to classify anemia morphologically, assess bone marrow response, evaluate iron status, and interpret ferritin in the context of inflammation. 3, 1

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Step 1: Classify by MCV and Interpret RDW

Microcytic Anemia (MCV <80 fL)

• Iron deficiency anemia is the most common cause worldwide, but do not assume this diagnosis without confirmatory iron studies. 4, 1
• An elevated RDW with microcytosis strongly suggests iron deficiency, even when MCV remains borderline normal. 3, 1
• Low MCH with elevated RDW is highly sensitive (72%) for detecting iron-deficient erythropoiesis before frank anemia develops. 1

Common pitfall: Mixed deficiencies (iron plus B12 or folate) can neutralize each other, yielding a normal MCV but elevated RDW—always check iron studies even when MCV appears normal. 3, 1, 2

Normocytic Anemia (MCV 80–100 fL)

• This pattern suggests hemorrhage, hemolysis, anemia of chronic disease, or early nutritional deficiency. 1, 2
• The reticulocyte count is critical here: it distinguishes decreased RBC production (low/normal reticulocyte index <2.0) from increased destruction or loss (elevated reticulocyte index >2.0). 2, 1

Macrocytic Anemia (MCV >100 fL)

• Vitamin B12 or folate deficiency is the primary consideration, but also evaluate for hypothyroidism, liver disease, alcohol use, and myelodysplastic syndrome. 1, 5
• A low RDW (<14%) with macrocytosis indicates a uniform population of enlarged cells, pointing toward megaloblastic anemia or thiopurine therapy rather than iron deficiency. 1, 5

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Step 2: Use Reticulocyte Count to Direct Further Testing

Low or Normal Reticulocyte Count (Inadequate Marrow Response)

This indicates impaired erythropoiesis; proceed with the following based on MCV: 1, 2

• Microcytic: Confirm iron deficiency with ferritin and transferrin saturation; if normal, obtain hemoglobin electrophoresis to exclude thalassemia trait. 3, 1
• Normocytic: Measure serum creatinine and eGFR (chronic kidney disease), vitamin B12, folate, TSH (hypothyroidism), and review medications. 1, 2
• Macrocytic: Order vitamin B12 and red blood cell folate levels immediately; review peripheral smear for hypersegmented neutrophils (megaloblastic) or oval macrocytes. 1, 5

Elevated Reticulocyte Count (Appropriate Marrow Response)

This suggests hemolysis or acute blood loss; immediately evaluate for: 1, 2

• Hemolysis markers: Low haptoglobin, elevated LDH, elevated indirect bilirubin, and peripheral smear for schistocytes or spherocytes. 3, 1, 2
• Direct antiglobulin (Coombs) test to identify autoimmune hemolytic anemia. 1, 2
• Clinical bleeding: Stool guaiac for occult GI blood loss, assess for melena, hematochezia, menorrhagia, or hematuria. 1, 2

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Step 3: Interpret Iron Studies in Context of Inflammation

Without Inflammation (Normal CRP)

• Serum ferritin <30 µg/L confirms iron deficiency. 3, 1
• Transferrin saturation <15–20% supports iron deficiency and is less affected by inflammation than ferritin. 1, 5

With Inflammation (Elevated CRP)

• Ferritin can be falsely elevated as an acute-phase reactant; use transferrin saturation <15–20% as your primary indicator of iron deficiency. 3, 5, 2
• In inflammatory bowel disease or chronic disease, ferritin up to 100 µg/L may still be consistent with iron deficiency. 3, 1

Critical pitfall: Relying on ferritin alone in the presence of inflammation, chronic disease, malignancy, or liver disease will miss iron deficiency—always pair ferritin with transferrin saturation and CRP. 1, 5, 2

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Step 4: Targeted Testing for Specific Etiologies

Suspected Vitamin Deficiency

• Measure serum vitamin B12 and folate levels when MCV is elevated or when peripheral smear shows hypersegmented neutrophils. 1, 5
• In elderly patients or those with inflammatory bowel disease, simultaneous iron and B12 deficiencies can mask each other—check both. 1, 5

Suspected Hemolysis

• Order haptoglobin, LDH, indirect bilirubin, and peripheral smear; perform a direct antiglobulin test if autoimmune hemolysis is suspected. 3, 1, 2
• If initial hemolysis workup is nondiagnostic, consider paroxysmal nocturnal hemoglobinuria flow cytometry and a DIC panel. 1

Suspected Chronic Kidney Disease

• Measure serum creatinine and eGFR in all patients with normocytic anemia and a low reticulocyte index. 1, 2
• In patients with creatinine ≥2 mg/dL and normocytic anemia without another identifiable cause, erythropoietin deficiency is the most likely etiology. 1
• Routine measurement of serum erythropoietin levels is not indicated. 1

Suspected Gastrointestinal Blood Loss

• All adult men and postmenopausal women with confirmed iron deficiency require endoscopic evaluation to exclude GI malignancy, regardless of anemia severity. 1
• Perform stool guaiac testing when iron deficiency is confirmed. 1, 2

Suspected Thalassemia Trait

• If iron studies are normal in a patient with microcytic anemia and appropriate ethnic background, obtain hemoglobin electrophoresis. 1, 5
• Do not give iron supplementation to patients with thalassemia trait; offer genetic counseling if planning pregnancy. 1

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Step 5: Extended Workup for Unclear Cases

If the cause remains unclear after initial testing, consider: 5, 2

• Soluble transferrin receptor and percentage of hypochromic red cells to evaluate for functional iron deficiency. 5, 2
• Bone marrow aspiration and biopsy with hematology consultation if pancytopenia is present or myelodysplastic syndrome is suspected in elderly patients. 1, 5, 2
• Thyroid-stimulating hormone and liver function tests to rule out hypothyroidism and liver disease as reversible causes. 1, 5, 2

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Common Diagnostic Pitfalls to Avoid

• Do not rely on MCV alone: 16% of microcytic patients and 90% of macrocytic patients may have etiologies inconsistent with traditional MCV-guided classification. 6
• Do not skip peripheral smear review: Visual examination reveals findings not apparent from automated indices, such as hypersegmented neutrophils, schistocytes, or spherocytes. 5, 2, 7
• Do not interpret ferritin without CRP: Inflammation falsely elevates ferritin and will cause you to miss iron deficiency. 3, 1, 2
• Do not overlook combined deficiencies: Iron plus B12 or folate deficiency can neutralize MCV but produce an elevated RDW—check both iron and vitamin studies. 3, 1, 2

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Practical Diagnostic Algorithm Summary

1. Confirm anemia with hemoglobin thresholds and order CBC with indices, reticulocyte count, ferritin, transferrin saturation, and CRP. 1, 2
2. Classify by MCV (micro-, normo-, macrocytic) and note RDW. 3, 1
3. Use reticulocyte count to distinguish impaired production (low/normal) from hemolysis or bleeding (elevated). 1, 2
4. Interpret iron studies in the context of inflammation using CRP. 3, 1, 2
5. Order targeted tests based on MCV and reticulocyte pattern: 1, 5, 2
   • Microcytic + low reticulocyte → iron studies, then hemoglobin electrophoresis if normal
   • Normocytic + low reticulocyte → renal function, B12, folate, TSH
   • Macrocytic + low reticulocyte → B12, folate, peripheral smear, TSH, liver function
   • Any MCV + elevated reticulocyte → hemolysis panel (haptoglobin, LDH, bilirubin, Coombs) and assess for bleeding
6. Investigate underlying cause: GI evaluation for iron deficiency in adults, medication review for macrocytosis, renal function for normocytic anemia. 1, 5, 2
7. Refer to hematology for bone marrow examination if pancytopenia, unexplained cytopenias, or suspected myelodysplastic syndrome. 1, 5, 2