CLINICAL APPLICATION OF ANTIHUMANGLOBULIN
In routine blood group immunohematology, antihumanglobulin is used to detect, the coating of antibody molecules on the red cell in-vivo as in haemilytic disease of new born and autoimmune haemilytic anaemia (AIHA) or transfusion reaction due to blood group incompatibility. Antihumanglobulin can be used to detect incomplete antibody in blood circulation as in the case of immunization of pregnant lady, AHG can also be used to detect antibodies to leucocytes and platelets, antibodies which are complement fixing and antihumanglobulin can be use in research oriented problem in immunohaematology.
The clinical application of antihumanglobulin is based on 2 two methods namely: Direct Coombs test also known as direct antihumaglobuin test (DAT) and indirect Coombs test also known as indirect antiglobulin test (IAT).
Direct Coombs Test and it’s Clinical Uses
This is a test used to detect antibodies or complement system factors that have bound to red blood cell surface antigens in vivo. It is not required for pre-transfusion testing but may be included by some laboratories.
The direct Coombs test is used clinically, when immune-mediated haemolytic anaemia (antibody-mediated destruction of red blood cells) is suspected. A positive Coombs test indicates that an immune mechanism is attacking the patient’s own red blood cells. This mechanism could be autoimmunity, alloimmunity or a drug-induced immune-mediated mechanism.
Example of alloimmune heamolysis
- Haemolytic disease of the newborn.
- Alloimmune haemolytic transfusion reactions
Example of autoimmune haemolysis
- Warm antibody autoimmune haemolytic anaemia
- Cold antibody immunohemolytic anaemia
ANTIHUMANGLOBULIN: Drug-induced immune-mediated haemolysis
- Methyldopa (lgG mediated type II hypersensitivity)
- Penicillin (high dose)
- Quindine (lgG mediated activation of classic complement pathway membrance attack complex (MAC)
In direct Coombs test, the patients red blood cells are washed and then incubated with antihuman globulin also known as Coombs reagent. If immunoglobulin or complement factors have been fixed onto the red blood cell surface in-vivo, the antihumanglobulin will facilitate agglutination of the red blood cells and the direct Coombs test will be positive.
Diseases that give a Positive Direct Coombs Test
1. Haemolytic disease of the new born is a disease condition that gives rise to positive direct Coombs test. It is a condition in which transplacental passage of maternal antibodies could be naturally occurring (anti A, and B) or immune antibodies which develop following a sensitizing event like transfusion or pregnancy. The hemolytic process may result in anaemia or hyperbilirubinemia or both; thereby affecting fetal or neonatal morbidity and mortality.
In HDN, foetal red blood cells in vivo are sensitized with lgG alloantibody of maternal origin thereby demonstrating a positive DAT with cord red blood cells. The most commonly observed HDN is due to Rho(D) incompatibility between mother and foetus.
2. A patient with transfusion reactions will demonstrate positive DAT, if serum contains antibodies against red blood cell antigens of donor red blood cells. Likewise antibody present in donor’s plasma, may also react with recipient red blood cells thereby demonstrating positive DAT.
- In drug induced haemolytic anaemia, certain drugs namely penicillin, procainamide, cephalosporins may also be associated with immune red blood cell destruction thereby demonstrating a positive DAT. Drug-induced hemolytic anaemias involve various theoretical mechanisms of activity and account for 12%-18% of autoimmunehaemolytic anaemia cases. (Wright et al., 1999, Gehrs et al., 2002). Classifications have involved immune versus non-immune or have centered on the proposed activity thought to cause the ultimate haemolysis. However, the AABB has also presented referenced theories that the antibodies may form against the drug entirely, a combination of the RBC membrane and drug membrane components, or mainly against the RBC membrane (Regardless of the a positive direct antiglobulin) test (DAT), hemolytic anaemia caused by drug therapies are rare. (Wright 1999).
- Alloimmune haemolytic anaemias occurs from alloantibodies that were immunologically derived secondary to a sensitizing event. ABO antibodies, however, are naturally occurring and can provide the most severe acute hemolytic episode, if matched with the wrong donor type. For other antibody types, a sensitizing event is on in which a patient has been exposed to red blood cell antigens that they lack. Sensitizing events can include blood exposure through transfusion, pregnancy, and intravenous drug use.
- Another disease that gives rise to positive DAT is autoimmune haemolytic anaemia (AIHA) which is characterized by an increased breakdown of red blood cells due to autoantibodies with or without complement activation. The diagnostic features of AIHA include the combination of clinical and laboratory signs of red blood cell hemolysis together with the detection of autoantibodies and or complement deposition on red blood cell as mostly evidence by a positive direct antiglobulin test (DAT).
In more than 50% of the patient the development of AHIA is associated with an underlying disease but can occur without any evidence of an underlying disorder. (Engelfriet et al., 1987). Based on the optimal temperature for autoantibody binding to red blood cell, AIHA is divided into a warm antibody AIHA cold antibody AIHA or AIHA due to biphasic auto antibody. A positive direct antiglobulin test (DAT) is an important finding in the diagnosis of autoimmune haemolytic anaemia. However, red blood cell antibodies of the immunoglobulin A class are not common and most of the laboratories use only anti-human lgG and C3d reagents for routine DAT. Therefore, the detection of lgA autoantibodies as a causative antibody AIHA is difficult.
Nevertheless, Agglutination of the red blood cells with the direct antiglobulin test is a positive result and indicates an auto-agglutinin or auto-antibodies while no agglytination is a negative result and indicates the absence of an unexpected antibody or lack of an auto-agglutinin.
ANTIHUMANGLOBULIN: Indirect Coombs Test and it’s Clinical Uses
It is a test used to detect in-vitro antibody-antigen reactions. It is used to detect very low concentrations of antibodies present in a patient’s plasma or serum prior to a blood transfusion. In antenatal care, the IAT is used to screen pregnant women for antibodies that may cause hemolytic disease of the newborn. The IAT can also be used for compatibility testing, antibody identification, red blood cell phenotyping and titration studies.
Examples of clinical uses of the indirect Coombs test include.
- Blood transfusion preparation in which the indirect Coombs test is used to screen for antibodies in the preparation of blood for blood transfusion. The donor’s and recipient blood must be ABO and Rh D compatible. Donor blood for transfusion is also screened for infections in separate processes.
- Antenatal antibody screening, the Coombs test is also used to screen pregnant women for lgG antibodies that are likely to pass through the placenta into the fetal blood and cause haemolytic disease of the newborn.
The purpose of the indirect antiglobulin test is to detect in-vitro sensitization of red cells. This is done when sensitization does not lead to direct agglutination. This occurs when there are too few antigens on the red cell, too few antibodies in the serum and those antibodies are in the lgG class.
Therefore agglutination of the red blood cells with the indirect antiglobulin test is a positive result and indicates the presence of unexpected antibodies.