Hemolytic anemias

Diagnostic Hallmarks of Hemolysis

• ↑ Reticulocyte count (RPI >2.5): appropriate marrow response.
• ↑ LDH: non-specific but sensitive.
• ↑ Indirect bilirubin (unconjugated; bili usually <5 unless concomitant Gilbert or liver disease).
• ↓ Haptoglobin (binds free Hb; depleted in intravascular hemolysis; less sensitive in extravascular alone).
• Hemoglobinuria, hemosiderinuria: intravascular hemolysis.
• Smear: schistocytes (mechanical or MAHA), spherocytes (HS or AIHA), bite/blister cells (G6PD), sickle cells, target cells, basophilic stippling (pyrimidine 5'-nucleotidase deficiency, lead).

Intravascular vs Extravascular Hemolysis

• Intravascular: complement-mediated lysis or mechanical fragmentation. Free Hb saturates haptoglobin scavenging, causing hemoglobinuria, hemosiderinuria, very low haptoglobin, and methemalbuminemia. Examples: PNH, mechanical valves, MAHA (TTP, HUS, DIC), severe G6PD crisis, acute hemolytic transfusion reaction (ABO-incompatible), march hemoglobinuria.
• Extravascular: RBCs cleared by macrophages in spleen and liver. Less marked ↑ LDH, more prominent ↑ indirect bili. Examples: warm AIHA, hereditary spherocytosis, sickle cell, mild G6PD.

Classification by Mechanism

• Inherited, membrane: hereditary spherocytosis (ankyrin, spectrin, band 3, protein 4.2), hereditary elliptocytosis (spectrin), hereditary stomatocytosis/xerocytosis. See note on hereditary hemolytic anemias.
• Inherited, enzyme: G6PD deficiency (X-linked, oxidative crisis), pyruvate kinase deficiency (chronic non-spherocytic), pyrimidine 5'-nucleotidase deficiency.
• Inherited, hemoglobin: SCD, thalassemia, HbC, HbE, unstable hemoglobins.
• Acquired, immune (Coombs positive): warm AIHA (IgG; idiopathic, lupus, CLL, drug-induced), cold agglutinin disease (CAD, primary clonal, monoclonal IgM); cold agglutinin syndrome (CAS) when secondary to Mycoplasma, EBV, or lymphoma/CLL, paroxysmal cold hemoglobinuria (Donath-Landsteiner, post-viral children). See note on AIHA.
• Acquired, non-immune (Coombs negative): PNH (acquired GPI-anchor deficiency, complement-mediated), MAHA (TTP, HUS, DIC, HELLP, malignant HTN, scleroderma renal crisis), prosthetic valve, malaria/babesia, snake or spider bite, Clostridium sepsis, Wilson disease, severe burns.

The DAT (Direct Antiglobulin Test, Coombs)

• Premise: do RBCs carry surface-bound IgG and/or complement (C3)? This categorizes hemolysis as immune-dependent or immune-independent.
• Principle: antihuman globulin (AHG) agglutinates antibody-coated cells. Standard testing uses polyspecific AHG containing both anti-IgG and anti-complement (anti-C3d).
• Sensitivity: detects roughly 100 to 500 molecules of IgG or 400 to 1,100 molecules of C3d per RBC. Fewer than 100 molecules can still cause clinically significant hemolysis, so a negative DAT does not exclude hemolysis; 5 to 10% of AIHA is DAT-negative. Misses IgA and IgM (and low-affinity IgG).
• Caveats: DAT strength correlates poorly with severity of hemolysis. A positive DAT does not by itself mean hemolysis is occurring: 1:1000 to 1:14,000 healthy donors and 7 to 8% of hospitalized patients have a positive DAT.

Initial Workup Algorithm

• Confirm hemolysis: retic count, LDH, indirect bili, haptoglobin, smear.
• DAT (Coombs):
  • Positive: immune-mediated; IgG +/- C3d = warm AIHA, C3d-only = CAD/CAS, PCH, or some warm/drug-induced; also exclude alloimmune/transfusion and drug causes. Work up secondary causes (lupus, CLL, lymphoma, drug, infection, immunodeficiency).
  • Negative: think non-immune. Congenital (HS, G6PD, SCD), MAHA, PNH, mechanical, infection, drug.
• If MAHA pattern: ADAMTS13 (TTP if <10%), Coombs, peripheral smear, BUN/Cr, platelets, fibrinogen, D-dimer, troponin.
• If suspect PNH: flow cytometry for CD55/CD59 deficiency on RBCs and granulocytes (FLAER on granulocytes is most sensitive).
• If suspect G6PD: a low level during crisis still confirms deficiency, but a normal/borderline result may be falsely reassuring (reticulocytosis); repeat 2 to 3 months after recovery if suspicion persists.
• Hb electrophoresis/HPLC: SCD, thalassemia, HbC.
• Osmotic fragility or EMA binding test: hereditary spherocytosis.
• Thick and thin smear: malaria, babesia.

High-Yield Pearls

• Step 1 to differentiate: DAT, positive vs negative.
• Spherocytes plus DAT positive = warm AIHA; spherocytes plus DAT negative suggests hereditary spherocytosis (confirm with EMA binding and family history; DAT-negative AIHA can mimic).
• Bite/blister cells plus Heinz bodies = G6PD oxidative hemolysis (post-fava, sulfa, primaquine, dapsone, infection).
• Schistocytes plus thrombocytopenia: emergency, exclude TTP (ADAMTS13).
• Cold agglutinin titer plus Mycoplasma or EBV in a young patient with post-infectious hemolysis.
• PNH: pancytopenia plus thrombosis (Budd-Chiari, splanchnic) plus dark morning urine; C5 inhibitors (eculizumab, ravulizumab, crovalimab), proximal inhibitors pegcetacoplan (C3) or iptacopan (oral factor B).
• Warm AIHA: 1L steroids, then 2L rituximab, then 3L splenectomy or fostamatinib (Tavalisse, SYK inhibitor; FDA Apr 17, 2018 for chronic ITP, used off-label for AIHA; phase 2 SOAR response 46%, but phase 3 FORWARD missed its primary endpoint, 35.6% vs 26.7%, p=0.398) or azathioprine/MMF.
• Interpret G6PD testing during a crisis with caution: a low level confirms deficiency, but a normal result can be falsely reassuring because reticulocytes have higher enzyme levels; repeat 2 to 3 months after recovery.
• A positive DAT does not equal hemolysis, and a negative DAT does not exclude it (5 to 10% of AIHA is DAT-negative).