Antiglobulin Test Coombs Test The antiglobulin test The

Antiglobulin Test (Coomb’s Test)

The antiglobulin test • The antiglobulin test was first introduced into clinical medicine in 1945 by R. R. Coombs • He showed that it could be used to detect nonagglutinating red cell antibodies or sensitized red cells • Most non-agglutinating (incomplete) antibodies are Ig. G, • These antibodies do not spontaneously cause agglutination due to a strong electronegative charge on the red cell surface that prevents the cells from coming into close proximity. • The antiglobulin reagent is able to bridge these negative forces.

Antihuman Globulin Definition: • Antihuman: antibodies against human antigens • Globulin: all antibody molecules are globulins Therefore: Antihuman Globulin is antibody directed against the Fc portion of human antibodies and/or complement components.

AHG Technique: What is the importance? • Some very clinically significant unexpected antibodies (eg. Kidd, Duffy) attach to red cell but do NOT cause agglutination at immediate spin or 37 o phase. • Yet, these antibodies are capable of causing severe hemolytic transfusion reactions or hemolytic disease of the newborn.

ANTIGLOBULIN TEST • Detection of non-agglutinating Abs, especially Ig. G or complement components (C 3) attached to RBCs in vivo or in vitro. – Direct antiglobulin test (DAT) - detection of sensitization of RBC s (coated with Abs and/or complement components) in vivo – Indirect antiglobulin test (IDAT) - detection of sensitization of RBCs (coated with Abs and/or complement components) in vitro

Antiglobulin Test • Principle - Antihuman globulins (AHG) from immunized animals bind to human globulins either free in serum or attached to RBCs – Pentameric Ig. M Abs are so large that, when bound to RBC Ags, the RBCs agglutinate (usually at RT) – Ig. G Abs usually need a little help, a bridge molecule, to agglutinate RBCs – AHG acts as a bridge molecule

Reagent Preparation • Polyclonal or monoclonal sources – Polyclonal - Animals immunized with human globulins (Ig. G & complement (C 3); bled for antisera to obtain high titered – Monoclonal - Hybridoma cells

Reagent Preparation • Polyspecific AHG – Abs to human Ig. G, and – Abs to human C 3 d (C 3 b breaks down to C 3 c and C 3 d) – Advantage is that polyspecific AHG may detect complement-dependent Abs on RBCs – Disadvantage - more nuisance positives • Monospecific AHG – Abs to human Ig. G only or human C 3 d only – Fewer nuisance positives; may miss an important Ab

Antiglobulin Test

Direct Antiglobulin Test • Detects in vivo sensitization of RBCs • The DAT is used to detect Ig. G or C 3 bound to the surface of the red cell. • In patients with hemolysis, the DAT is useful in determining whethere is an – Immune etiology – OR Non-immune etiology of hemolysis

Direct Antiglobulin Test

Non-immune causes of hemolysis • Mechanical hemolysis such as those due to artificial valves or burns, • Hemoglobinopathies (sickle cell, thalassemia), • Red cell enzyme deficiencies (G 6 PDP, pyruvate kinase), • and red cell membrane defects (hereditary spherocytosis, PNH) Have a negative DAT

DAT Uses § Immune causes of hemolysis § § Autoimmune hemolytic anemias, Drug induced hemolysis, Delayed or acute hemolytic transfusion Hemolytic Disease of the new born Have a positive DAT

Indirect Antiglobulin Test (IAT) • Detection of in vitro sensitization of RBCs • Most clinically significant alloantibodies are Ig. G antibodies that react best at 37 o. C and are formed as a result of previous exposure via transfusion or pregnancy. • Examples include antibodies to Rh, Kell, Kidd, and Duffy red cell antigens. – Same as DAT, except: – Step 1 entails incubating RBCs (reagent or unknown) with antisera (reagent or unknown) allowing time for in vitro attachment of Abs to RBCs

Indirect Antiglobulin Test

IAT Uses • When the unknown is sera: – Ab Screening: The patient’s serum is incubated with red cells of known phenotype to detect Ab in patient’s serum against a specific red cell Ag – Phenotyping: Ab of known specificity is incubated with patient’s RBC to identify specific blood group Ag on the cells – Crossmatch: The patient’s serum is incubated with donor red cells to detect Abs that might reduce the survival of transfused red cells

Modifications of IAT • Modifications can be done to increase sensitivity & to decrease time to perform IAT – Modification of suspending media – Modification of RBCs • Each modification has advantages and disadvantages

Modification of suspending medium Mechanism of action Advantages Long incubation time required Saline Not applicable expensive LISS Facilitates Short incubation sensitization but time diminishes agglutination Facilitates Short incubation sensitization and time, enhances agglutination certain clinically significant Abs Enhances the reaction caused by cold autoantibodies Albumin Inexpensive Disadvantages

Modification of RBCs • RBCs used in the IAT can be treated with enzymes • This increases sensitivity of test for detection of certain auto & allo-antibodies including Rhesus & Kidd • Enzyme treatment destroy certain Abs including M, N, S & Duffy

Importance of Anti-complement in antiglobulin reagents • Anticomplement increases detection sensitivity for certain Abs (Anti-Kidd) • Approx. 200 molecules of cell bound Ig. G required for +ve antiglobulin test • Ab sensitization below this level will not be detected by anti-Ig. G alone • If antiglobulin reagent contains both anti. Ig. G & anti-C 3 b will increase probability of detection of sensitization

False Positive Reactions False Positive Mechanism Cold Autoantibody “in vitro” complement fixation & auto-agglutination Technical Dirty tubes, colloidal silica Anti-species Ab Heterologous Ab in the antiglobulin reagent Polyagglutinable red cells Presence of anti-T or anti-Tn in the antiglobulin reagent

False Negative Failure to add reagent Technical problems Neutralization of the antiglobulin reagent due to insufficient washing of the test cells Contamination or neutralization of antiglobulin reagent False –ve test can be detected by addition of Ig. G sensitized cells to –ve antiglobulin tests. These cells should be agglutinated by the anti-Ig. G