Which laboratory test is most useful to differentiate iron‑deficiency microcytic anemia from thalassemia trait?

Laboratory Test to Distinguish Iron Deficiency Anemia from Thalassemia Trait

The combination of serum ferritin and red blood cell distribution width (RDW) provides the most useful discrimination: serum ferritin <30 μg/L with RDW >14% indicates iron deficiency anemia, whereas normal/elevated ferritin with RDW ≤14% suggests thalassemia trait. 1, 2, 3

Primary Screening Tests

Serum ferritin is the single most powerful and cost-effective test for distinguishing these conditions:

• Ferritin <15 μg/L is 99% specific for iron deficiency and essentially rules out thalassemia trait 1, 2
• Ferritin <30 μg/L indicates depleted iron stores consistent with iron deficiency 1, 2
• Normal or elevated ferritin (>30 μg/L) in the presence of microcytosis strongly suggests thalassemia trait rather than iron deficiency 4, 5, 6

Red blood cell distribution width (RDW) provides critical additional discrimination:

• RDW >14% combined with low MCV indicates iron deficiency anemia, reflecting the heterogeneous population of red cells (older normal-sized cells mixed with newer microcytic cells) 1, 2, 3
• RDW ≤14% with low MCV suggests thalassemia trait, where red cells are uniformly small 1, 2, 3
• Approximately 50% of thalassemia cases may show elevated RDW, so this finding does not definitively exclude thalassemia but makes iron deficiency more likely 3

Confirmatory Iron Studies When Ferritin Is Equivocal

If ferritin levels are borderline (30-100 μg/L) or potentially elevated by inflammation, add transferrin saturation:

• Transferrin saturation <16-20% confirms iron deficiency even when ferritin appears falsely normal due to inflammation 1, 2, 3
• Calculate as: (serum iron ÷ total iron-binding capacity) × 100 2, 3
• In thalassemia trait, transferrin saturation is typically normal or elevated 7, 5

Total iron-binding capacity (TIBC) provides supporting evidence:

• Elevated TIBC (>400 μg/dL) indicates iron deficiency 2, 8
• Normal or low TIBC suggests thalassemia trait or anemia of chronic disease 7, 5

When to Order Hemoglobin Electrophoresis

Hemoglobin electrophoresis should be reserved for specific scenarios and is not a first-line test:

• Order when microcytosis persists with normal iron studies (ferritin >30 μg/L, transferrin saturation >20%) 1, 3, 7
• Order when MCV is disproportionately low relative to the degree of anemia 1, 3
• Order in patients of African, Mediterranean, or Southeast Asian ancestry with microcytosis and normal ferritin 3, 7
• Order when anemia fails to improve after 4 weeks of adequate oral iron therapy despite documented compliance 3
• Beta-thalassemia trait shows elevated hemoglobin A2 levels (>3.5%) on electrophoresis 7

Mean Corpuscular Hemoglobin (MCH) as an Alternative Index

MCH may be more reliable than MCV for detecting iron deficiency:

• MCH is less dependent on specimen storage conditions and laboratory equipment variability 2, 3
• MCH is reduced in both absolute and functional iron deficiency 2
• Low MCH combined with low mean corpuscular hemoglobin concentration (MCHC) strongly suggests hypochromic anemia from iron deficiency 3

Practical Diagnostic Algorithm

Step 1: Order serum ferritin and complete blood count with RDW as initial tests 1, 2, 7, 6

Step 2: Interpret the combination:

• Ferritin <30 μg/L + RDW >14% = Iron deficiency anemia (proceed to identify bleeding source) 1, 2, 3
• Ferritin >30 μg/L + RDW ≤14% = Thalassemia trait (confirm with hemoglobin electrophoresis) 3, 7

Step 3: If ferritin is 30-100 μg/L (equivocal range), add:

• Transferrin saturation: <16-20% confirms iron deficiency 1, 2, 3
• C-reactive protein to assess for inflammation that may falsely elevate ferritin 1, 3

Step 4: Order hemoglobin electrophoresis only if iron studies are normal/borderline or patient has appropriate ethnic background 1, 3, 7

Critical Pitfalls to Avoid

Do not assume all microcytic anemia is iron deficiency—thalassemia trait, anemia of chronic disease, and sideroblastic anemia require different management and unnecessary iron therapy can be harmful 3, 8, 7

Do not order hemoglobin electrophoresis as a first-line test—it is expensive and should be reserved for cases where iron studies are normal or borderline 1, 3

Do not rely on serum iron alone—it has significant day-to-day variation and overlaps considerably between iron deficiency and thalassemia trait 1, 4, 5

Recognize that ferritin can be falsely elevated by inflammation, infection, malignancy, or liver disease—in these settings, transferrin saturation <20% helps confirm true iron deficiency 1, 2, 3

Consider combined deficiencies—iron deficiency can coexist with folate or vitamin B12 deficiency, which may also produce an elevated RDW 3