What is the recommended work-up for a patient suspected of having thalassemia (a genetic disorder affecting hemoglobin production) versus a hemoglobinopathy (a group of disorders affecting hemoglobin structure or function)?

Diagnostic Work-up for Thalassemia versus Hemoglobinopathy

Begin with a complete blood count (CBC) with mean corpuscular volume (MCV), red cell distribution width (RDW), and reticulocyte count, followed by iron studies (ferritin, serum iron, TIBC, transferrin saturation) to exclude iron deficiency, then proceed to hemoglobin analysis via high-performance liquid chromatography (HPLC) or hemoglobin electrophoresis to differentiate thalassemia from structural hemoglobinopathies, and confirm with molecular genetic testing when indicated. 1, 2, 3

Initial Laboratory Assessment

Complete Blood Count Analysis

• Obtain CBC with specific attention to MCV, RDW, and red blood cell count. 1 Thalassemia trait typically presents with microcytosis (low MCV) but an elevated or normal red blood cell count despite low hemoglobin, distinguishing it from iron deficiency which shows low red cell count. 1
• Measure RDW to help differentiate causes of microcytosis. 1 An elevated RDW suggests iron deficiency or combined pathology, while a normal RDW is more consistent with thalassemia trait. 1
• Check reticulocyte count to assess for hemolysis or ineffective erythropoiesis. 1

Iron Studies to Exclude Iron Deficiency

• Measure serum ferritin as the most powerful test for iron deficiency, with levels <12 μg/dL diagnostic in the absence of inflammation. 1 In the presence of inflammation, ferritin up to 100 μg/L may still indicate iron deficiency. 1
• Obtain serum iron, total iron binding capacity (TIBC), and transferrin saturation, with <16% saturation suggesting iron deficiency. 1
• Measure C-reactive protein (CRP) to assess for inflammation that may elevate ferritin falsely. 1

Critical pitfall: Do not assume microcytosis in certain ethnic groups (Mediterranean, Southeast Asian, African, Middle Eastern descent) is due to iron deficiency without confirming with laboratory testing, as hemoglobinopathies are common in these populations. 1, 4, 2

Hemoglobin Analysis

When to Proceed to Hemoglobin Studies

• If iron studies are normal but microcytosis persists, proceed to hemoglobin analysis to evaluate for thalassemia or hemoglobinopathy. 1, 3
• If there is a family history of anemia, transfusion dependence, or known hemoglobinopathy, proceed directly to hemoglobin analysis regardless of iron studies. 5, 3

Hemoglobin Electrophoresis/HPLC

• Perform high-performance liquid chromatography (HPLC) or hemoglobin electrophoresis as the primary method to detect abnormal hemoglobin variants and quantify hemoglobin fractions (HbA, HbA2, HbF, HbS, HbC, HbE). 2, 3, 6
• Elevated HbA2 (>3.5%) confirms β-thalassemia trait in the setting of microcytosis and normal iron studies. 5, 3
• Presence of abnormal hemoglobin peaks (HbS, HbC, HbE, HbD) indicates structural hemoglobinopathy. 2, 3
• Elevated HbF (>1% in adults) may suggest β-thalassemia, hereditary persistence of fetal hemoglobin, or certain hemoglobinopathies. 3

Important distinction: Thalassemia is caused by quantitative defects (reduced production of normal globin chains), while hemoglobinopathies involve structural defects (abnormal amino acid sequences in globin chains). 2, 3

Molecular Genetic Testing

Indications for Genetic Testing

• Confirm diagnosis when HPLC/electrophoresis results are equivocal or show patterns consistent with α-thalassemia (which may have normal HbA2 and HbF). 2, 3
• Identify specific mutations in α-globin or β-globin genes using DNA analysis, particularly when considering prenatal diagnosis or family screening. 2, 3
• Use massively parallel sequencing (next-generation sequencing) for comprehensive mutation detection when standard methods fail to identify the defect. 2

Specific Genetic Patterns

• α-thalassemia is most commonly caused by deletions in α-globin genes, requiring molecular testing for definitive diagnosis as hemoglobin analysis may be normal or show only mild abnormalities. 2, 3
• β-thalassemia is associated with point mutations in β-globin gene, which can be identified by targeted sequencing. 2, 3

Differentiating Thalassemia from Hemoglobinopathy

Thalassemia Features

• Microcytosis with elevated or normal red blood cell count despite anemia. 1, 3
• Normal or elevated HbA2 (β-thalassemia trait) or normal hemoglobin pattern with genetic confirmation (α-thalassemia). 3
• Family history often reveals similar findings in parents or siblings. 3

Hemoglobinopathy Features

• Presence of abnormal hemoglobin variants (HbS, HbC, HbE, HbD, HbM) on HPLC/electrophoresis. 2, 3
• Variable MCV depending on the specific hemoglobinopathy; may be normocytic or microcytic. 3
• Clinical manifestations vary widely: HbSS (sickle cell disease) causes vaso-occlusive crises and hemolysis; HbE/β-thalassemia causes transfusion-dependent anemia; HbM causes methemoglobinemia with cyanosis. 5, 3

Combined Disorders

Concurrent Thalassemia and Iron Deficiency

• A therapeutic trial of iron supplementation for 3 weeks may help confirm true iron deficiency, with monitoring of hemoglobin response. 1 If hemoglobin rises significantly, iron deficiency was present; if minimal response, thalassemia trait is the primary cause of microcytosis. 1
• Initiate oral iron supplementation while investigating the underlying cause of iron deficiency (gastrointestinal blood loss, malabsorption, dietary insufficiency). 1
• Consider adding ascorbic acid to improve iron absorption in resistant cases. 1

Combined Hemoglobinopathies

• Compound heterozygous states (e.g., HbS/β-thalassemia, HbE/β-thalassemia) require both hemoglobin analysis and genetic testing for accurate diagnosis. 3
• Clinical severity varies: HbE/β-thalassemia can range from mild anemia to transfusion-dependent disease. 3

Family Screening and Genetic Counseling

• Test first-degree relatives of patients with confirmed hereditary thalassemia or hemoglobinopathy to identify carriers and provide genetic counseling. 5
• Prenatal diagnosis using genetic testing enables prevention of severe thalassemia births and management of pregnancy complications. 2
• Inform patients about autosomal recessive inheritance for thalassemia and autosomal dominant inheritance for some hemoglobinopathies (HbM disease). 5, 3

Follow-up and Monitoring

• For confirmed iron deficiency (with or without thalassemia trait), monitor hemoglobin and iron studies after 4-8 weeks of iron therapy. 1
• Investigate the underlying cause of iron deficiency, particularly gastrointestinal sources of blood loss in adults. 1
• For diagnosed thalassemia or hemoglobinopathy, refer to hematology for comprehensive management, including assessment for transfusion dependence, iron chelation needs, and monitoring for organ complications. 7, 3