direct Coombs

**Principle:** The patient's washed RBCs are mixed with Coombs reagent (anti-human globulin, which contains anti-IgG and/or anti-C3). If IgG or complement is already coated on the RBCs, the reagent cross-links the cells and causes visible agglutination, which is a positive test.

**Direct vs. indirect Coombs:** - Direct (DAT) tests for antibodies or complement bound to the patient's RBCs in vivo. It uses the patient's RBCs plus Coombs reagent. - Indirect (IAT) tests for free antibodies in the patient's serum. Patient serum is incubated with reagent RBCs, then Coombs reagent is added. It is used for prenatal screening and for pretransfusion antibody screening and crossmatching. - Memory aid: Direct = the problem is on the patient's cells now. Indirect = look for antibodies floating in serum that could cause a future problem.

**Positive DAT – key causes:** 1. **Warm AIHA** (IgG, extravascular hemolysis in the spleen). Associations include SLE, CLL, lymphomas, and drugs such as methyldopa. Treatment is steroids first-line, then rituximab or splenectomy. The DAT is positive for IgG ± C3. 2. **Cold AIHA / cold agglutinin disease** (IgM, which fixes complement). Associations include Mycoplasma pneumoniae, EBV/infectious mononucleosis, and lymphoproliferative disorders. The DAT is positive for C3 only, because IgM falls off at warmer temperatures. Hemolysis is mostly extravascular, with some intravascular. Patients have acrocyanosis in the cold, and the smear shows RBC clumping. 3. **Hemolytic disease of the newborn.** This includes Rh incompatibility (Rh-negative mother, Rh-positive fetus) and ABO incompatibility (type O mother, type A or B infant). Maternal IgG crosses the placenta. The infant's DAT is positive, usually more strongly in Rh disease and often weakly positive or even negative in ABO disease. 4. **Transfusion reactions.** Acute or delayed hemolytic transfusion reactions give a positive DAT. Delayed reactions are anamnestic responses to minor antigens such as Kidd, Rh, or Kell. 5. **Drug-induced immune hemolysis.** Mechanisms include hapten (penicillin, cephalosporins), immune complex ("innocent bystander," e.g., quinidine), and autoantibody induction (methyldopa).

**Lab findings in hemolysis (with a positive DAT):** Anemia, ↑ reticulocytes, ↑ indirect bilirubin, ↑ LDH, ↓ haptoglobin, and spherocytes on smear (extravascular, IgG-mediated). Intravascular hemolysis also causes hemoglobinuria and hemosiderinuria.

**Negative DAT hemolysis (high-yield contrast):** Hereditary spherocytosis (positive osmotic fragility test or EMA binding), G6PD deficiency (Heinz and bite cells), PNH (flow cytometry for CD55/CD59), microangiopathic hemolysis (TTP/HUS/DIC with schistocytes), and mechanical valve hemolysis.

**Type II hypersensitivity link:** AIHA, hemolytic disease of the newborn, and transfusion reactions are type II (antibody-mediated) hypersensitivity, and the DAT is the classic test for this.

**Management pearl:** Treat the underlying cause. Use steroids for warm AIHA. For cold AIHA, keep the patient warm and consider rituximab. Rh-negative mothers receive RhoGAM (anti-D IgG) at about 28 weeks and postpartum to prevent sensitization.