Anemia is defined as a reduction in the oxygen-carrying capacity of blood, reflected by low hemoglobin, hematocrit, or RBC count below the age- and sex-adjusted reference range. The laboratory approach to anemia classification uses two complementary frameworks: the morphologic classification based on RBC indices, and the etiologic (pathophysiologic) classification based on bone marrow response measured by the reticulocyte count.
Morphologic Classification by MCV
The mean corpuscular volume (MCV) divides anemias into three categories. Microcytic anemia (MCV < 80 fL): iron deficiency anemia is the most common cause worldwide; other causes include thalassemia trait, anemia of chronic disease (sometimes normocytic), sideroblastic anemia (can be microcytic or dimorphic), and lead poisoning. The RDW helps distinguish iron deficiency (elevated RDW, anisocytosis) from thalassemia trait (normal RDW). Normocytic anemia (MCV 80–100 fL): acute blood loss, hemolytic anemias, anemia of chronic disease, aplastic anemia, myelodysplastic syndromes, bone marrow infiltration, renal failure (decreased erythropoietin), and mixed deficiency. Macrocytic anemia (MCV > 100 fL): megaloblastic anemias (B12 deficiency, folate deficiency) with elevated RDW and hypersegmented neutrophils; non-megaloblastic causes include liver disease, alcohol use, hypothyroidism, myelodysplasia, and reticulocytosis (large reticulocytes raise MCV).
Etiologic Classification by Reticulocyte Response
The reticulocyte production index (RPI) splits anemias into two categories. Hyperproliferative anemia (RPI > 2.5–3.0): the bone marrow is responding appropriately to anemia by increasing RBC production, indicating that the problem is peripheral RBC loss or destruction. This category includes acute blood loss (hemorrhage) and hemolytic anemias (immune, hereditary, mechanical, enzyme deficiency, hemoglobinopathy). Hypoproliferative anemia (RPI < 2.0): the bone marrow is not producing enough RBCs for the degree of anemia. This category includes nutritional deficiencies (iron, B12, folate), anemia of chronic disease/inflammation, bone marrow failure (aplastic anemia, myelodysplasia, infiltration), and renal failure (erythropoietin deficiency).
Systematic Approach to Anemia Workup
The laboratory workup of anemia proceeds in a stepwise fashion. Step 1: Confirm anemia on the CBC and review RBC indices (MCV, MCH, MCHC, RDW). Step 2: Examine the peripheral blood smear for RBC morphology (size, shape, color, inclusions), WBC abnormalities, and platelet assessment. Step 3: Determine the reticulocyte count and calculate RPI to categorize as hyper- or hypoproliferative. Step 4: Based on MCV and RPI, order targeted tests — iron studies (ferritin, TIBC, transferrin saturation) for microcytic hypoproliferative; B12, folate, and MMA/homocysteine for macrocytic hypoproliferative; hemolysis markers (LDH, haptoglobin, bilirubin, DAT) for hyperproliferative with evidence of hemolysis. Step 5: If the initial workup is unrevealing, consider bone marrow aspiration and biopsy, hemoglobin electrophoresis, flow cytometry, or cytogenetic/molecular testing.
Iron Studies in Microcytic Anemia
Serum ferritin is the most specific test for iron deficiency; a low ferritin (< 15–30 ng/mL) is diagnostic of iron deficiency. Ferritin is an acute-phase reactant and can be falsely normal in inflammation. Total iron-binding capacity (TIBC) is elevated in iron deficiency and low in inflammation. Transferrin saturation (serum iron ÷ TIBC × 100) below 16% suggests iron-deficient erythropoiesis. Soluble transferrin receptor (sTfR) is elevated in iron deficiency and unaffected by inflammation, making it useful for distinguishing iron deficiency from anemia of chronic disease. MCV and RDW are discussed in detail in the RBC indices article.
Macrocytic Anemia Workup
Vitamin B12 and folate levels are the first-line tests. B12 deficiency can be due to pernicious anemia (autoimmune, anti-intrinsic factor antibodies), malabsorption (gastric bypass, Crohn disease, celiac disease, pancreatic insufficiency), dietary deficiency (vegan diet), or proton pump inhibitor use. Methylmalonic acid (MMA) and homocysteine are more sensitive markers for B12 deficiency. In folate deficiency, homocysteine is elevated but MMA is normal. The peripheral blood smear shows macro-ovalocytes and hypersegmented neutrophils (≥ 5 lobes). Bone marrow aspiration reveals megaloblastic changes (nuclear-cytoplasmic dyssynchrony) in erythroid precursors.
Hemolytic Anemia Evaluation
When the RPI is elevated and hemolysis is suspected, initial testing includes: LDH (elevated), haptoglobin (decreased), indirect bilirubin (elevated), and urinalysis for hemoglobinuria. The direct antiglobulin test (DAT, Coombs test) identifies antibody- or complement-coated RBCs, confirming autoimmune hemolytic anemia. The peripheral blood smear is examined for spherocytes (AIHA, hereditary spherocytosis), schistocytes (microangiopathic hemolytic anemia), sickle cells, or bite cells (G6PD deficiency). Further testing is guided by smear and DAT results: hemoglobin electrophoresis for hemoglobinopathies, RBC enzyme assays for G6PD/pyruvate kinase deficiency, and osmotic fragility test for hereditary spherocytosis.
