What are the non‑immune and non‑PNH causes of hemolytic anemia when the direct antiglobulin (Coombs) test and the paroxysmal nocturnal hemoglobinuria (PNH) panel are negative?

Non-Immune, Non-PNH Causes of Hemolytic Anemia

When both the Coombs test and PNH panel are negative, the primary causes to investigate are hereditary red cell defects (membranopathies, enzymopathies, hemoglobinopathies), thrombotic microangiopathies, drug-induced hemolysis, and Wilson's disease—with Wilson's disease being the most critical emergency to exclude in acute presentations. 1

Immediate Life-Threatening Causes to Exclude

Wilson's Disease (Most Critical)

• Wilson's disease presenting with acute liver failure and Coombs-negative hemolytic anemia requires urgent liver transplantation and carries 95% mortality if untreated. 1
• Suspect Wilson's disease when acute liver failure combines with Coombs-negative intravascular hemolysis, modest aminotransferase elevations, low alkaline phosphatase, and alkaline phosphatase-to-bilirubin ratio <2. 1
• Key diagnostic features include serum copper ≥200 μg/dL, coagulopathy unresponsive to vitamin K, and rapid progression to hepatic/renal failure. 1
• Kayser-Fleischer rings may be absent in 50% of acute presentations, so their absence does not exclude the diagnosis. 1
• Measure serum ceruloplasmin, serum copper, 24-hour urinary copper, and perform slit-lamp examination immediately. 1

Thrombotic Microangiopathy (TMA)

• The presence of schistocytes on peripheral blood smear indicates microangiopathic hemolytic anemia and suggests TMA rather than autoimmune causes. 2
• Check platelet count and renal function urgently—thrombocytopenia (<150,000/mm³ or 25% reduction) plus renal involvement defines atypical hemolytic uremic syndrome (aHUS). 2
• Measure ADAMTS13 activity emergently to exclude thrombotic thrombocytopenic purpura (TTP); severely deficient activity indicates TTP, not aHUS. 2
• If aHUS is confirmed, initiate urgent complement inhibition therapy (eculizumab) rather than corticosteroids. 2

Drug-Induced Hemolysis

• Evaluate for common drug causes including dapsone, primaquine, ribavirin, rifampin, interferon, cephalosporins, penicillins, NSAIDs, quinine/quinidine, fludarabine, ciprofloxacin, and diclofenac. 3
• Dapsone and primaquine cause dose-related hemolysis, particularly in G6PD deficiency, through oxidative mechanisms. 4, 5
• Obtain detailed medication history including recent antibiotic exposures and over-the-counter medications. 3

Systematic Diagnostic Approach

Essential Initial Laboratory Tests

• Peripheral blood smear examination is mandatory—it differentiates mechanical hemolysis (schistocytes) from corpuscular defects (spherocytes, target cells, sickle cells). 2, 6
• Confirm hemolysis with haptoglobin (most sensitive), LDH, and unconjugated bilirubin. 6
• Reticulocyte count should exceed 120×10⁹/L unless vitamin deficiency is present or hemolysis is very acute. 6
• Check platelet count and renal function to evaluate for TMA. 1, 2

Second-Line Specialized Testing

Once emergencies are excluded, pursue these tests systematically: 6

For Hereditary Causes:

• Glucose-6-phosphate dehydrogenase (G6PD) assay—the most common enzymopathy causing hemolysis. 3, 4
• Hemoglobin phenotyping to detect hemoglobinopathies (sickle cell disease, thalassemia, hemoglobin C, unstable hemoglobins). 6
• Eosin-5'-maleimide (EMA) binding test for membranopathies (hereditary spherocytosis, stomatocytosis). 6
• Additional enzyme assays if G6PD is normal (pyruvate kinase deficiency, other rare enzymopathies). 6

For Metabolic/Toxic Causes:

• Assess for methemoglobinemia. 3
• Consider Zieve's syndrome in alcoholism context. 6
• Evaluate for spur cell anemia in advanced cirrhosis. 6

Hereditary Red Cell Defects

Membranopathies

• Hereditary spherocytosis and stomatocytosis cause chronic hemolysis with spherocytes on smear and negative Coombs test. 6
• EMA binding test is the diagnostic standard for membranopathies. 6

Enzymopathies

• G6PD deficiency is the most common cause—hemolysis is triggered by oxidative stress from infections, drugs, or fava beans. 4, 5
• G6PD testing must be performed before prescribing primaquine or dapsone due to severe hemolytic risk. 4, 5
• Pyruvate kinase deficiency and other rare enzyme defects require specialized enzymatic assays. 6

Hemoglobinopathies

• Sickle cell disease, thalassemia syndromes, hemoglobin C disease, and unstable hemoglobins all cause Coombs-negative hemolysis. 6
• Hemoglobin phenotyping identifies these disorders. 6

Additional Workup for Refractory Cases

Bone Marrow Failure Evaluation

If hemolysis is refractory or diagnosis remains unclear: 3

• Check vitamin B12, folate, copper, iron studies, and thyroid function. 3
• Test for parvovirus infection. 3
• Consider bone marrow biopsy with cytogenetic analysis to exclude myelodysplastic syndromes. 3

Infectious and Systemic Causes

• Evaluate for viral/bacterial causes including mycoplasma. 3
• Perform protein electrophoresis and cryoglobulin analysis. 3
• Consider autoimmune serology if systemic symptoms present. 3

Critical Clinical Pitfalls

Common Diagnostic Errors:

• Assuming negative Coombs test excludes all immune causes—rare cases of PNH can be Coombs-positive, requiring comprehensive hemolysis workup. 7
• Missing Wilson's disease in young patients with acute hemolysis—this is uniformly fatal without transplantation. 1
• Failing to check peripheral smear before initiating corticosteroids—schistocytes indicate TMA requiring complement inhibition, not steroids. 2
• Overlooking drug history—many common medications cause hemolysis in susceptible individuals. 4, 5

Testing Limitations:

• G6PD testing may be falsely normal during acute hemolysis due to reticulocytosis (young cells have higher enzyme levels)—retest after recovery if suspicion remains. 4, 5
• Small PNH clones can be missed by older assays—latest generation flow cytometry detects very small populations. 8