What is the mechanism of the Coombs test?

Mechanism of the Coombs Test

The Coombs test detects the presence of antibodies or complement bound to red blood cells, allowing for the diagnosis of immune-mediated hemolytic conditions through an antigen-antibody reaction. 1

Types of Coombs Tests

• Direct Coombs Test (Direct Antiglobulin Test - DAT):

  • Detects immunoglobulin, complement, or both already bound to the patient's red blood cell membrane 2
  • Used to diagnose autoimmune hemolytic anemia, hemolytic disease of the newborn, and drug-induced hemolytic anemia 1
  • A negative direct Coombs test is normal in healthy individuals and rules out classic autoimmune hemolytic anemia 3

• Indirect Coombs Test (Indirect Antiglobulin Test - IAT):

  • Detects circulating antibodies in serum that can bind to red blood cells 4
  • Used for antibody screening before blood transfusions, detecting maternal antibodies in pregnancy, and identifying alloimmunization 4, 5

Mechanism of Action

• Direct Coombs Test:

  • Patient's red blood cells are washed to remove unbound serum proteins 1
  • Anti-human globulin reagent (Coombs reagent) is added to the washed cells 1
  • If immunoglobulins or complement are present on RBC surfaces, the anti-human globulin binds to them 6
  • Visible agglutination occurs, indicating a positive result 1

• Indirect Coombs Test:

  • Patient's serum is incubated with known test red blood cells 4
  • If antibodies in the serum bind to antigens on the test cells, they will attach to the RBC surface 4
  • After washing to remove unbound antibodies, anti-human globulin reagent is added 4
  • Agglutination indicates antibodies in the patient's serum bound to the test cells 4, 5

Clinical Applications

• Autoimmune Hemolytic Anemia (AIHA):

  • Direct Coombs test is positive in warm-antibody AIHA 4
  • Helps distinguish immune from non-immune causes of hemolysis 3

• Hemolytic Disease of the Newborn:

  • Direct Coombs test on newborn's RBCs detects maternal antibodies bound to fetal cells 2
  • Indirect Coombs test on maternal serum identifies circulating antibodies that could affect the fetus 5

• Transfusion Medicine:

  • Used in pre-transfusion testing to detect antibodies that could cause transfusion reactions 6
  • Helps investigate suspected transfusion reactions 6

• Cancer Patients:

  • Recommended for patients with chronic lymphocytic leukemia and non-Hodgkin lymphoma before initiating erythropoiesis-stimulating agents 7

Reagents Used

• Polyspecific Coombs Reagent:

  • Contains antibodies against human IgG, IgM, and complement component C3 8
  • Detects multiple types of immunoglobulins and complement on RBCs 8

• Monospecific Coombs Reagent:

  • Contains antibodies against a single immunoglobulin class or complement component 8
  • Helps determine the specific type of antibody or complement involved 8

Potential Pitfalls

• False-Negative Results:

  • Insufficient washing of RBCs before adding anti-human globulin 1
  • Low density of antibodies on RBC surface 9
  • Prozone effect (excess antibody preventing agglutination) 9

• False-Positive Results:

  • Improper technique leading to RBC rouleaux formation 1
  • Contamination of reagents 8
  • Spontaneous agglutination of RBCs 1

• Enhanced Sensitivity Techniques:

  • Testing at different temperatures (4°C, 37°C) 9
  • Using increased dilutions of antiglobulin reagent 9
  • Flow cytometry for improved detection of RBC-bound immunoglobulin 9

Diagnostic Algorithms

• When Hemolysis is Suspected:

  • Perform complete blood count with reticulocyte count 4
  • Examine peripheral blood smear for morphological abnormalities 4
  • Check hemolysis markers (LDH, indirect bilirubin, haptoglobin) 3
  • Perform direct Coombs test to determine if hemolysis is immune-mediated 3
  • If direct Coombs is negative with evidence of hemolysis, consider mechanical causes like microangiopathic hemolytic anemia 3

• In Pregnancy with Positive Indirect Coombs:

  • Perform detailed ultrasound with fetal echocardiography 5
  • Evaluate middle cerebral artery Doppler for signs of fetal anemia 5
  • Consider amniocentesis for PCR testing of infectious agents if clinically indicated 5
  • Monitor for development of hydrops fetalis 5