LECTURE 11 (Agglutination & Coombs test)

Question 1

What is Coombs testing/Antiglobulin testing?

Answer 1

A test used to evaluate haemolytic anaemias and for pre-transfusion testing

DESCRIPTION:
COOMBS SERUM is added to patient’s blood which contain ANTIHUMAN GLOBULINS (AHGs) that detect + adhere with 2 binding sites to IMMUNE PROTEINS that mediate hemolysis and/or complement (e.g antibodies)

NEGATIVE RESULT -> no proteins are present so AHGs will not bind to anything

POSITIVE RESULT -> RBC agglutination
[proteins coat the RBC surface then serum is added which causes multiple RBC to adhere together]

Question 2

What is Direct Coombs test (DAT)?

Answer 2

The key test to diagnose haemolytic anemia

FUNCTION:
- detection of antibodies and/or complement proteins on the surface of RBCs
- narrow the differential of hemolysis into either ANTIBODY-MEDIATED HEMOLYSIS or NON-ANTIBODY MEDIATED HEMOLYSIS

INDICATIONS:
all patients with hemolysis

METHOD:
1) Patient’s blood sample is purified so that only RBCs remain
2) COOMBS SERUM is added + AHGs bind to patient’s antibodies and/or complement on surface of RBC -> agglutination
POSITIVE RESULT = indicates that the patient’s RBCs are coated with AUTOANTIBODIES and/or complement that are causing hemolysis
NEGATIVE RESULT = non-antibody mediated hemolysis

Question 3

What are the causes of a positive Direct Coombs test?

Answer 3

• Autoimmune hemolytic anemia
• Immune haemolytic anemia
  [Tranfusion reaction + Haemolytic disease of the newborn]
• Drug induced autoimmune haemolytic anemia
  [e.g penicillins, cephalosporin, immunotherapy, chemotherapy]

Question 4

What is Indirect Coombs test?

Answer 4

The test used to detect anti-RBC antibodies in the serum of transfusion recipients and patients with haemolytic disease of the foetus and newborn (HDFN)

INDICATIONS:
- any patient requiring pre-transfusion testing
- patients with suspected transfusion reactions
- newborns with signs of hemolysis
- screening for Rh-negative mothers to determine if anti-D immunoglobulin is indicated

METHOD:
1) Patient’s blood sample is purified so that only the serum remains
2) Patient’s serum is incubated with test RBCs -> If there are anti-RBC antibodies in the patient’s serum, it will bind to the test RBCs
3) Coombs serum is added + AHGs bind to the patient’s antibodies which are bound to the test RBCs -> RBC agglutination

Question 5

What is the difference between Direct Coombs test and Indirect Coombs test?

Answer 5

CLINICAL APPLICATIONS:
- DCT = workup of haemolytic anaemia
- ICT = prevention of transfusion reaction + prevention of HDFN (haemolytic disease of the foetus and newborn)

LOCATION OF ANTIBODIES DETECTED:
- DCT = RBC surface
- ICT = serum

COOMBS SERUM IS ADDED TO:
- DCT = patient’s purified RBCs
- ICT = patient’s purified serum which has been mixed with test RBCs

AHGs IN COOMBS SERUM BIND TO:
- DCT = antibodies and/or complement already coating the patient’s RBCs
- ICT = the patient’s antibodies which are bound to test RBCs

Question 6

What is Cold agglutinin haemolytic anaemia (cold AIHA)?

Answer 6

A group of autoimmune disorders characterised by hemolysis that is caused by the binding of cold-sensitive autoantibodies to RBCs

PATHOPHYSIOLOGY:
Cold-sensitive antibodies/cold agglutinins (mostly IgM antibodies) cause EXTRAVASCULAR HEMOLYSIS and ACUTE INTRAVASCULAR HEMOLYSIS -> antigen-antibody reaction is triggered by low body temp/cold environment
- STABLE DISEASE: extravascular hemolysis of complement-coated RBCs by the mononuclear phagocyte system
- ACUTE EXACERBATIONS: intravascular hemolysis can occur rarely, mediated through the complement system (activated by IgM antibodies bounds to RBCs)

CAUSES:
- idiopathic (unknown cause)
- can occur secondary to MYCOPLASMA PNEUMONIAE or EBV INFECTION or MALIGNANCY (e.g non-hodgkin lymphoma, CLL)

SYMPTOMS:
- pallor, fatigue, weakness
- painful cyanosis of extremities (acrocyanosis)
- raynaud phenomenon
- skin ulcerations

DIAGNOSIS:
- evidence of haemolytic anemia
- positive direct Coombs test for C3d

Question 7

What is warm agglutinin haemolytic anemia?

Answer 7

An autoimmune disease characterised by the binding of heat sensitive autoantibodies to RBCs which leads to the phagocytosis and destruction of RBCs in the reticuloendothelial system

PATHOPHYSIOLOGY:
Heat-sensitive antibodies (IgG) bind to multiple RBC antigens, then to Fc receptors on phagocytes (triggered by high body temp) -> binding of antibodies leads to increased EXTRAVASCULAR HEMOLYSIS mediated by the recituloendothelial system of the SPLEEN and LIVER

CAUSES:
- idiopathic (unknown cause)
- secondary causes: malignancy (lymphoma, chronic lymphocytic leukaemia), autoimmune diseases (SLE), certain drugs (rifampin, penicillins, phenytoin, a-methyldopa)

SYMPTOMS:
- pallor, fatigue, weakness
- mild splenomegaly

DIAGNOSIS:
- evidence of extravascular hemolysis
- positive direct Coombs test for IgG