What is the initial workup for a patient presenting with hemolytic anemia?

Initial Workup for Hemolytic Anemia

Begin with a complete blood count with red blood cell indices, peripheral blood smear, reticulocyte count, lactate dehydrogenase, haptoglobin, indirect bilirubin, and direct antiglobulin test as the essential first-line laboratory panel. 1, 2, 3

Essential First-Line Laboratory Tests

The initial diagnostic approach requires a systematic laboratory evaluation to confirm hemolysis and guide further workup:

• Complete blood count with red blood cell indices assesses anemia severity and identifies whether the anemia is normocytic or macrocytic, which should prompt consideration of hemolysis 1, 3

• Peripheral blood smear is mandatory to identify schistocytes (suggesting microangiopathic hemolysis), spherocytes (suggesting hereditary spherocytosis or autoimmune hemolytic anemia), or other morphologic abnormalities that narrow the differential diagnosis 1, 2, 3

• Reticulocyte count evaluates bone marrow compensatory response and is typically elevated in hemolysis, though it may be inappropriately low in 20-40% of autoimmune hemolytic anemia cases (a poor prognostic factor), or when marrow involvement, nutritional deficiency, parvovirus B19 infection, or autoimmune suppression is present 1, 4, 5

• Lactate dehydrogenase (LDH) is elevated as a marker of intravascular hemolysis and red cell destruction 1, 2, 4

• Haptoglobin is decreased due to binding of free hemoglobin released during hemolysis 1, 2, 4

• Indirect (unconjugated) bilirubin is elevated from red blood cell breakdown 1, 2, 4

• Direct antiglobulin test (Coombs test) with monospecific antisera is the cornerstone test that differentiates immune-mediated hemolytic anemia from non-immune causes 1, 2, 3, 4

Critical History and Physical Examination Elements

A focused history and examination identifies potential triggers and hereditary patterns:

• Drug exposure history must specifically query ribavirin, rifampin, dapsone, interferon, cephalosporins, penicillins, NSAIDs, quinine/quinidine, fludarabine, ciprofloxacin, lorazepam, and diclofenac 6, 1, 2

• Recent infections including mycoplasma, viral pathogens, or history of insect/spider/snake bites can trigger acute hemolysis 1, 2

• Family history of anemia, neonatal jaundice, early cholecystectomy, splenomegaly, or unexplained jaundice suggests hereditary hemolytic conditions such as hereditary spherocytosis, G6PD deficiency, or pyruvate kinase deficiency 1, 3

• Physical findings to document include jaundice, splenomegaly, pallor, dark urine, and signs of underlying autoimmune or lymphoproliferative disorders 1, 2

Second-Tier Diagnostic Tests Based on Initial Results

If Direct Antiglobulin Test is Positive (Immune-Mediated):

• Autoimmune serology panel evaluates for underlying systemic autoimmune disease such as systemic lupus erythematosus 1

• Protein electrophoresis and cryoglobulin analysis exclude lymphoproliferative disorders that can cause secondary autoimmune hemolytic anemia 1

If Direct Antiglobulin Test is Negative (Non-Immune):

• Glucose-6-phosphate dehydrogenase (G6PD) assay evaluates for the most common red cell enzyme deficiency, though testing should be delayed until after acute hemolysis resolves to avoid false-negative results 1, 3

• Flow cytometry for paroxysmal nocturnal hemoglobinuria (PNH) screening is necessary, as PNH presents with intravascular hemolysis, hemosiderinuria, and markedly elevated LDH 1, 4

• Hemoglobin electrophoresis evaluates for hemoglobinopathies including sickle cell disease and thalassemias 1, 3

If Microangiopathic Features Present on Smear:

• DIC panel including PT/INR, fibrinogen, and D-dimer distinguishes disseminated intravascular coagulation from thrombotic microangiopathies 1, 2

• ADAMTS13 activity level and inhibitor titer is mandatory if thrombotic thrombocytopenic purpura (TTP) is suspected, as delay in diagnosis increases mortality and morbidity; immediate hematology consultation and plasma exchange should be initiated while awaiting results 6, 1, 2

Workup for Refractory or Unclear Etiology

When the diagnosis remains elusive after initial testing:

• Bone marrow examination with cytogenetics evaluates for myelodysplastic syndromes, marrow failure syndromes, or infiltrative processes 1, 2

• Vitamin B12, folate, and copper levels exclude nutritional causes of inadequate reticulocyte response that may mask hemolysis 1

• Parvovirus B19 testing is necessary if reticulocytopenia is present, as this virus selectively infects red cell precursors 1

• Iron studies (ferritin, transferrin saturation) and thyroid function tests complete the comprehensive evaluation 1

• Methemoglobin level should be assessed if cyanosis or oxidative drug exposure is present 6, 1

• Pyruvate kinase activity assay should be considered in patients with chronic unexplained hemolytic anemia, particularly if there is a family history, neonatal hyperbilirubinemia, or increased reticulocytosis after splenectomy 6

Common Pitfalls to Avoid

• Do not assume adequate reticulocytosis rules out hemolysis—reticulocytopenia occurs in 20-40% of autoimmune hemolytic anemia cases and indicates poor prognosis 4, 7

• Avoid testing G6PD levels during acute hemolysis, as young reticulocytes have higher enzyme activity and may yield false-negative results 3

• Do not transfuse platelets empirically in suspected TTP, as this may worsen thrombosis; plasma exchange is the priority treatment 6, 8

• Recognize that warm autoimmune hemolytic anemia with complement-positive DAT, mixed-type AIHA, and atypical AIHA are associated with more severe disease, higher transfusion requirements, and increased mortality 7

• Consider that iron overload (elevated ferritin and transferrin saturation) may occur disproportionately to transfusion history in chronic hemolytic conditions 6