What is the differential diagnosis and treatment approach for hemolytic anemia?

Differential Diagnosis of Hemolytic Anemia

The differential diagnosis of hemolytic anemia requires systematic classification into immune versus non-immune causes, followed by specific testing to identify the underlying etiology, with the direct antiglobulin test (DAT) serving as the critical initial differentiating test. 1, 2

Initial Diagnostic Workup

When hemolytic anemia is suspected, confirm hemolysis with the following laboratory markers:

• Elevated reticulocyte count (indicates compensatory marrow response, though may be absent in 20-40% of autoimmune cases—a poor prognostic sign) 1, 3
• Elevated lactate dehydrogenase (LDH) (marker of intravascular hemolysis) 1, 2, 3
• Decreased or absent haptoglobin 1, 2, 3
• Elevated indirect (unconjugated) bilirubin 1, 2, 3
• Peripheral blood smear to identify red blood cell morphology abnormalities 1, 2
• Complete blood count showing normocytic or macrocytic anemia 1, 2

Important caveat: Reticulocytopenia occurs in 20-40% of autoimmune hemolytic anemia cases due to marrow involvement, nutritional deficiencies, infections, or autoimmune reaction against bone marrow precursors, and represents a poor prognostic factor. 3

Primary Classification: Immune vs. Non-Immune

Direct Antiglobulin Test (DAT/Coombs Test)

Perform the DAT before initiating any treatment—this is the cornerstone test that differentiates immune from non-immune hemolysis. 1, 2

• Monospecific DAT is mandatory in the diagnostic workup to identify the specific antibody type (IgG vs. complement) 4
• Indirect antiglobulin test detects free autoantibodies in serum 1

Critical pitfall: Autoantibody titers may vary during disease course; seronegative individuals at diagnosis may express autoantibodies later, requiring repeated testing if clinical suspicion remains high. 1

Differential Diagnosis Categories

If DAT Positive: Immune-Mediated Hemolytic Anemia

Autoimmune Hemolytic Anemia (AIHA)

• Warm antibody AIHA (IgG-mediated, most common type) 4
• Cold agglutinin disease (complement-mediated) 4
• Mixed type AIHA 4

Evaluate for secondary causes:

• Lymphoproliferative disorders 1
• Autoimmune diseases (perform comprehensive autoimmune serology) 1
• Infections (viral, bacterial including mycoplasma) 1
• Drug-induced hemolysis: ribavirin, rifampin, dapsone, interferon, cephalosporins, penicillins, NSAIDs, quinine/quinidine, fludarabine, ciprofloxacin, lorazepam, diclofenac 1

Alloimmune Hemolysis

• Transfusion reactions
• Hemolytic disease of the newborn

If DAT Negative: Non-Immune Hemolytic Anemia

Hereditary Causes

Membranopathies:

• Hereditary spherocytosis
• Hereditary elliptocytosis
• Dehydrated hereditary stomatocytosis (DHSt)—now recognized as the second most frequent congenital hemolytic anemia with availability of genetic testing 5

Enzymopathies:

• Pyruvate kinase (PK) deficiency: Most common glycolytic pathway enzyme defect and most common cause of hereditary nonspherocytic hemolytic anemia 6

  • Suspect in patients with chronic anemia, splenomegaly, jaundice with normal or near-normal RBC morphology 6
  • Transfusion-dependent cases of unknown etiology 6
  • Severe neonatal indirect hyperbilirubinemia 6
  • High reticulocyte count in splenectomized patients without diagnosis 6
  • Critical pitfall: False-normal PK enzyme activity occurs with markedly elevated reticulocytes, recent transfusions, or incomplete removal of platelets/leukocytes 1

• G6PD deficiency: Hemolysis triggered by oxidant drugs or infections 6, 1

  • Heinz bodies may be visible on peripheral smear 6
  • Isopropanol or heat stability testing altered 6

Hemoglobinopathies:

• Unstable hemoglobin variants: May present with hemolysis and elevated methemoglobin levels 6

  • Examples: HbChile (chronic methemoglobinemia), others develop methemoglobin only after stressor events 6

• M-hemoglobins: Autosomal dominant, present with cyanosis and hemolytic anemia (particularly HbM Saskatoon and HbM Hyde Park) 6

  • α-globin variants: cyanosis evident at birth 6
  • β-globin variants: manifest at 6-9 months when β chains replace fetal γ chains 6
  • Electrophoresis at pH 7.1 identifies HbM variants 6

• Cytochrome b5 reductase (CYB5R3) deficiency: Congenital methemoglobinemia with residual enzyme activity <20% of normal 6

  • Evaluate for consanguinity (more common in this deficiency) 6
  • Long-life history of cyanosis or family history of dusky skin/blue sclera suggests congenital forms 6

Acquired Non-Immune Causes

Thrombotic microangiopathies:

• Thrombotic thrombocytopenic purpura (TTP)
• Hemolytic uremic syndrome (HUS)
• HELLP syndrome
• Disseminated intravascular coagulation (DIC)
• Note: Thrombocytopenia suggests possible thrombotic microangiopathy 3

Mechanical trauma:

• Prosthetic heart valves (associated with intravascular and chronic hemolysis) 3
• Cardiac stenting 3
• March hemoglobinuria

Infections:

• Malaria
• Babesiosis
• Clostridial sepsis
• Bartonellosis

Oxidative damage:

• Evaluate for methemoglobinemia 1
• Chemical/toxin exposure

Paroxysmal Nocturnal Hemoglobinuria (PNH):

• Screen with flow cytometry 1
• Marked LDH elevation and hemosiderinuria typical of intravascular hemolysis 3

Hypersplenism and systemic diseases:

• Liver disease
• Renal disease

Advanced Diagnostic Testing

When Initial Workup is Non-Diagnostic

For suspected hereditary hemolytic anemia with unclear etiology:

• Next-generation sequencing (NGS) gene panel analyzing 68+ hemolytic anemia-related genes is now the gold standard for hereditary methemoglobinemia and congenital hemolytic anemias 6, 5
• Target-captured sequencing particularly useful for DHSt diagnosis 5
• Molecular testing enables confirmation of PK deficiency when enzyme assays are equivocal 6
• For PK deficiency genotype discrepancies: assays for large deletions, re-evaluation with specific tests or NGS platform 6

Additional specialized testing:

• Bone marrow analysis and cytogenetic analysis if myelodysplastic syndrome suspected 1
• HIV and hepatitis testing 1
• Protein electrophoresis and cryoglobulin analysis 1
• B12, folate, copper, parvovirus, iron studies, thyroid function if refractory 1

Important consideration: Comprehensive autoimmune serology may not be available in all laboratories; send samples to reference laboratories in cases of diagnostic uncertainty. 1

Treatment Approach Based on Etiology

Autoimmune Hemolytic Anemia (Warm Type)

First-line therapy: Corticosteroids (1 mg/kg methylprednisolone or equivalent) 7, 8, 4

• Prednisone is FDA-approved for acquired (autoimmune) hemolytic anemia 7

Second-line/severe cases:

• Add rituximab early in severe cases or if no prompt response to steroids 4
• Consider rituximab 375 mg/m² weekly 8
• IVIG may be added when rapid response required or fulminant hemolysis occurs 8

Refractory cases:

• Plasma exchange (5 sessions every other day) as bridge to splenectomy in severe, refractory anemia with cardiovascular compromise 8
• Splenectomy for definitive management 8
• Other immunosuppressives (cyclosporine, bendamustine) 8, 4

Cold Agglutinin Disease

First-line: Rituximab with or without bendamustine 4

Hereditary Causes

• PK deficiency: Monitor iron overload; gene therapy and activator treatments in development 6
• G6PD deficiency: Avoid oxidant triggers
• Membranopathies: Splenectomy may be indicated
• Unstable hemoglobins: Avoid oxidant stressors; supportive care

Acquired Non-Immune Causes

• TTP: Urgent plasma exchange
• Drug-induced: Discontinue offending agent
• Mechanical: Address underlying cardiac issue
• PNH: Complement inhibitors (eculizumab, ravulizumab)