Which laboratory tests should be ordered to evaluate anemia?

Laboratory Tests for Anemia Evaluation

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 the essential first-line panel for any patient presenting with anemia. 1

Core Initial Laboratory Panel

The minimum diagnostic workup must include:

• Complete blood count (CBC) with red cell indices to assess hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and red cell distribution width (RDW). 1, 2

• Absolute reticulocyte count to distinguish impaired bone marrow production (low/normal count) from hemolysis or acute blood loss (elevated count). 1, 3

• Serum ferritin as the single most powerful test for iron deficiency, with levels <30 μg/L confirming depleted iron stores and <15 μg/L providing 99% specificity for iron deficiency. 1, 4

• Transferrin saturation (TSAT) calculated as serum iron × 100 ÷ total iron-binding capacity, with values <16–20% confirming iron deficiency and being less affected by inflammation than ferritin. 1, 4

• C-reactive protein (CRP) to identify inflammatory states that can falsely elevate ferritin, since ferritin is an acute-phase reactant. 1, 4

Algorithm Based on Red Cell Indices

Microcytic Anemia (MCV <80 fL)

When MCV is low, the differential diagnosis narrows significantly:

• If RDW >14%: Iron deficiency anemia is most likely, as the mixed population of older normal-sized cells and newer microcytic cells creates heterogeneity. 1, 4

• If RDW ≤14%: Thalassemia trait is more likely, as red cells are uniformly small. 1, 4

• Confirm with iron studies: Ferritin <30 μg/L and TSAT <20% establish iron deficiency; if iron studies are normal, proceed to hemoglobin electrophoresis to exclude thalassemia. 1, 4

• Order stool guaiac testing when iron deficiency is confirmed to screen for gastrointestinal bleeding. 5, 1

Normocytic Anemia (MCV 80–100 fL)

The reticulocyte count becomes the critical decision point:

• If reticulocyte index ≤2: Indicates inadequate bone marrow response; order vitamin B12, folate, thyroid-stimulating hormone (TSH), and assess renal function (creatinine/eGFR) to evaluate for chronic kidney disease. 5, 1

• If reticulocyte index >2–3: Indicates increased red cell production; order hemolysis panel including haptoglobin, lactate dehydrogenase (LDH), indirect bilirubin, and peripheral blood smear for schistocytes. 1, 3

• Measure iron studies even with normal MCV because early iron deficiency or anemia of chronic disease can present as normocytic. 1

Macrocytic Anemia (MCV >100 fL)

Macrocytosis requires evaluation for megaloblastic causes:

• If reticulocyte count is low/normal: Order serum vitamin B12 and folate levels to confirm or exclude megaloblastic anemia. 1

• If reticulocyte count is elevated: Proceed with hemolysis evaluation (haptoglobin, LDH, indirect bilirubin). 1

• Order TSH and liver function tests as hypothyroidism and liver disease are common reversible causes of macrocytic anemia. 1

• Check iron studies concurrently because combined iron and B12/folate deficiency can mask each other, normalizing the MCV but elevating the RDW. 1

Interpretation of Ferritin in Inflammatory States

Ferritin interpretation requires clinical context because inflammation falsely elevates this acute-phase reactant:

• Ferritin <30 μg/L: Confirms iron deficiency regardless of inflammatory status. 1, 4

• Ferritin 30–100 μg/L with elevated CRP: May still represent true iron deficiency; a TSAT <20% confirms iron-deficient erythropoiesis. 1, 4

• Ferritin >100 μg/L with TSAT <20% and elevated CRP: Defines anemia of chronic disease with functional iron deficiency. 1, 4

• Ferritin >150 μg/L: Makes absolute iron deficiency unlikely even in inflammatory conditions. 1

Additional Testing Based on Clinical Context

When Iron Studies Are Normal but Microcytosis Persists

• Order hemoglobin electrophoresis to screen for thalassemia trait or other hemoglobinopathies, particularly in patients of African, Mediterranean, or Southeast Asian ancestry. 1, 4

When Hemolysis Is Suspected

• Measure haptoglobin (low), LDH (elevated), and indirect bilirubin (elevated). 1

• Examine peripheral blood smear for schistocytes, spherocytes, or other morphologic abnormalities. 1, 6

• Perform direct antiglobulin (Coombs) test to identify autoimmune hemolytic anemia. 1

When Chronic Kidney Disease Is Present

• Assess serum creatinine and estimated glomerular filtration rate (eGFR). 5

• If creatinine ≥2 mg/dL and anemia is normocytic/normochromic with no other identified cause, erythropoietin deficiency is the likely primary etiology. 5

• Measurement of serum erythropoietin levels is usually not indicated in chronic kidney disease patients. 5

Critical Pitfalls to Avoid

• Do not rely on ferritin alone when CRP is elevated—always calculate TSAT to uncover hidden iron deficiency, as ferritin can be falsely normal (30–100 μg/L) despite true depletion. 1, 4

• Do not consider a "normal" reticulocyte count reassuring in anemic patients—it represents an inappropriately low marrow response and warrants evaluation for nutritional deficiencies or bone marrow failure. 1, 3

• Do not assume all microcytic anemia is iron deficiency—anemia of chronic disease, thalassemia, and sideroblastic anemia require different management and can be distinguished by iron studies and RDW. 1, 4

• Do not overlook combined deficiencies—iron deficiency can coexist with vitamin B12 or folate deficiency, recognizable by an elevated RDW despite a normal or near-normal MCV. 1

• Do not order hemoglobin electrophoresis as a first-line test—it should be reserved for cases with normal iron studies, extreme microcytosis disproportionate to anemia severity, or appropriate ethnic background. 1, 4

Monitoring Treatment Response

• Hemoglobin should rise ≥10 g/L within 2 weeks of starting oral iron therapy if iron deficiency is the cause; this response confirms the diagnosis even when initial iron studies were equivocal. 1, 4

• Reticulocyte count typically increases within 7–10 days of initiating appropriate therapy for nutritional deficiencies. 2

• Repeat iron studies 2–4 weeks after treating acute blood loss because iron deficiency may develop once stores become depleted. 1