Chapter 05 The Antiglobulin Test

The Antiglobulin Test

• The antiglobulin test (AGT), also known as the Coombs' test, detects antibodies (IgG) or complement attached to red blood cells (RBCs).
• AHGs obtained from immunized nonhuman species bind to human globulins like IgG or complement, whether free in serum or attached to antigens on RBCs.
• The AGT is essential in transfusion medicine, impacting patient well-being

History of the Antiglobulin Test

• Before the AGT, only IgM antibodies were detected.
• The introduction of the AGT permitted the detection of nonagglutinating IgG antibodies and led to the discovery and characterization of many new blood group systems.
• In 1945, Coombs and associates described the use of the AGT for detecting weak and nonagglutinating Rh antibodies in serum.
• In 1946, Coombs and coworkers described the use of AHG to detect in vivo sensitization of RBCs (later called the direct antiglobulin test [DAT]) of babies suffering from hemolytic disease of the newborn (HDN).
• Moreschi described the test's principle in 1908, using rabbit antigoat serum to agglutinate rabbit RBCs sensitized with low doses of goat antirabbit RBC serum.
• Coombs' procedure involved injecting human serum into rabbits to produce antihuman serum.
• In 1947, Coombs and Mourant demonstrated that the antibody activity that detected Rh antibodies was associated with the anti-gamma globulin fraction in the reagent.
• In 1951, Dacie presented the indication that another antibody activity influenced the final reaction; different reaction patterns were obtained when dilutions of AHG were used to test cells sensitized with warm as compared with cold antibodies.
• In 1957, Dacie and coworkers published data showing that the reactivity of AHG to cells sensitized with warm antibodies resulted from anti-gamma globulin activity, whereas anti-nongamma globulin activity was responsible for the activity of cells sensitized by cold antibodies.
• The antiglobulin test is used to detect RBCs sensitized with IgG alloantibodies, IgG autoantibodies, and complement components and can occur in vivo or in vitro.
• The indirect antiglobulin test (IAT) is a two-stage technique for detecting in vitro sensitization of RBCs.
• The direct antiglobulin test (DAT) is a one-stage procedure detecting in vivo sensitization.

Antihuman Globulin Reagents

• The Food and Drug Administration (FDA) Center for Biologics Evaluation and Research (CBER) defines several AHG reagents which can be polyspecific or monospecific.
• Polyspecific AHG reagents contain antibody to human IgG and to the C3d component of human complement and facilitate agglutination when RBCs have been sensitized with IgG or C3d or both.
• Monospecific AHG reagents contain only one antibody specificity, either anti-IgG or antibody to specific complement components such as C3b or C3d (i.e., anticomplement).
• Anti-IgG reagents contain antibodies specific for the Fc fragment of the gamma heavy chain of the IgG molecule.
• Anticomplement reagents, such as anti-C3b, anti-C3d reagents, are reactive against only the designated complement components and contain no activity against human immunoglobulins.
• The classic method of AHG production involves injecting human serum or purified globulin into laboratory animals such as rabbits.
• Human IgG injected into a rabbit results in anti-IgG production; human complement components injected into a rabbit result in anticomplement.
• This produces a polyclonal antiglobulin serum, a mixture of antibodies from different plasma cell clones.
• Monoclonal antibodies are derived from one clone of plasma cells and recognize a single epitope, produced using Hybridoma technology

Polyspecific AHG

• Polyspecific AHG can be made using polyclonal or monoclonal antibodies, but the two types of antibody production processes are very different from one another.
• Polyclonal AHG is usually prepared in rabbits and modern production commences with purifying the immunogen from a pool of normal sera.
• Conventional polyspecific antiglobulin reagents are produced by immunizing one colony of rabbits with human immunoglobulin (IgG) antigen and another colony with human C3 antigen.
• Blood specimens are drawn from the immunized animals and if the antibody potency and specificity meet predetermined specifications, the animals are bled for a production batch of reagent.
• Separate blends of the anti-IgG and anticomplement antibodies are made and each pool is absorbed with A1, B, and O cells to remove heterospecific antibodies.
• Antibody content of each pool is determined and the potency of the pools is analyzed to calculate optimum antibody dilution for use.
• For anti-IgG pools, block titrations are performed by reacting dilutions of each antibody against cells sensitized with variable amounts of IgG
• Potency of anti-C3 pools measured using at least two examples each of a C3b- and C3d-coated cell

Monoclonal AHG Production

• The monoclonal antibody technique devised by Kohler and Milstein has been used to produce AHG and has proved useful in producing high-titer antibodies with well-defined specificities to IgG and to the fragments of C3.
• Begins with immunization of animals (usually mice) with purified human globulin and antibody-secreting lymphocytes from mouse spleens which are fused with myeloma cells after a suitable immune response
• Resulting "hybridomas" are screened for antibodies with the required specificity and affinity and antibody-secreting clones propagated in tissue culture or by inoculation into mice
• Because the clonal line produces a single antibody, there is no need for absorption to remove heterospecific antibodies.
• Monoclonal antibody production: antibody with the same specificity and reaction characteristics will be available indefinitely.
• All antibodies produced by a clone of cells recognize a single epitope present on an antigen.
• Monoclonal antibodies to human complement components anti-C3b and anti-C3d may be blended with polyclonal anti-IgG from rabbits for more potent reagents that give fewer false-positive reactions

Monospecific AHG

• Monospecific AHG contains only one antibody specificity and is produced as a monoclonal, polyclonal, or blended formula.

Antibodies Required in AHG

• AHG must contain antibody activity to nonagglutinating blood group antibodies, mostly a mixture of IgG₁ and IgG3 subclasses.
• AHG must contain antibody activity to nonagglutinating blood group antibodies.
• Rarely, nonagglutinating IgM antibodies are found; they have always been shown to fix complement and may be detected by anticomplement and anti-IgM activity.
• Therefore, anti-IgG activity must be present, anti-IgM and anti-IgA activity may be present, but neither is essential.
• The presence of anti-light chain activity allows detection of all immunoglobulin classes.

Anticomplement

• Some antibodies "fix" complement components to the RBC membrane after complexing of the antibody with its corresponding antigen.
• After antibodies complex with corresponding antigens, complement is bound to RBC membrane
• The terms most, some, and rare refer to antibodies that bind complement the vast majority of the time, that show variability in their ability to bind complement, and that rarely bind complement, respectively.
• Membrane-bound complement components are detected by the anticomplement activity in AHG and as a result of studies published during the 1960s indicated the need for anticomplement activity in AHG to allow the IAT to detect antibodies,
• Presence of anticomplement activity enhances the reactions of clinically significant antibodies.
• When RBCs are incubated with serum for longer than 15 minutes, the number of C3c determinants falls rapidly because C3c is split off the C3bi molecule so the Joint Working Party supports the use of anti-C3d in international reference reagents. which can occur in vivo and is a common occurrence in both warm and old autoimmune hemolytic anemias.
• Garratty and Petz confirmed the need for anti-C3d activity in AHG for use in the DAT.

Use of Polyspecific Versus Monospecific AHG in the IAT

• Polyspecific AHG contains both anti-IgG activity and anti-C3 activity.
• Considerable debate on using monospecific anti-IgG versus polyspecific AHG for routine antibody detection and pretransfusion testing.
• Detecting IgG antibodies is the most important function of polyspecific AHG and there have been reports of clinically significant RBC alloantibodies that were undetectable with monospecific anti-IgG, but were detected with the anticomplement component of AHG.
• Polyspecific AHG associated with unwanted positive reactions from clinically insignificant antibodies
• Some clinically significant antibodies are detected with the anticomplement component of AHG but not with anti-IgG, especially true for anti-Jka
• Evaluating weak positive AHG reactions includes repeating with prewarmed technique, although not recommended, because about 60% of false-positive weak reactions become negative.
• Howard and associates found eight patients with Jka or Jkb specificity antibodies detected primarily by or solely with AHG containing anticomplement activity
• Complement-only Kidd antibodies represented 23% of all Kidd antibodies detected and the authors use polyspecific AHG reagent for routine compatibility testing.
• A decision for which AHG reagent to use is at the discretion of the individual blood bank and many use monospecific cost containment measures for pretransfusion, especially for anti-Jka

Principles of the Antiglobulin Test

• The antiglobulin test is based on these principles:
• Antibody molecules and complement components are globulins.
• Injecting an animal with human globulin stimulates antibody production to the foreign protein (i.e., AHG).
• Serologic tests employ a variety of AHG reagents (anti-IgG, anti-C3d, and polyspecific reagents containing both).
• AHG reacts with human globulin molecules, either bound to RBCs or free in serum.
• Washed RBCs coated with human globulin are agglutinated by AHG.

Direct Antiglobulin Test

• The direct antiglobulin test (DAT) detects in vivo sensitization of RBCs with IgG or complement components.
• The DAT is not a required test in routine pretransfusion protocols.
• Initial DATs include testing one drop of washed 3% to 5% suspension RBCs with polyspecific (anti-IgG, anti-C3d) reagent and positive results are monitored with DAT panel using monospecific anti-IgG and anti-C3d to determine protein sensitizing cell.
• The saline control serves to detect spontaneous agglutination of cells or reactions occurring without the addition of AHG reagents.
• In transfusion reactions, it detects the presence of IgG or C3

The Antihuman Globulin Test (AHG)

• 5-3(A) - Indirect AHG (IAT) with sensitization occuring invitro, looking for unkown antibody in patient's serum/plasma
• 5-3(b) - Direct AHG (DAT) with sensitization already occuring In Vivo
• Table 5-4 Depicts the DAT panel and patterns of reactivity in Autoimmune Hemolytic Anemia (AIHA)
• Clinical consideration dictates the degree of patient DAT evaluation required

Indirect Antiglobulin Test

• The IAT determines In Vitro RBC sensitization and is used in these situations:
• Detection of incomplete antibodies to potential donor RBC's
• Determination of RBC phenotype using known antisera
• Titration of incomplete antibodies

Factors Affecting the Antiglobulin Test

• The DAT can detect between 100 - 500 IgG molecules per RBC and 400-1100 molecules of C3d per RBC
• The IAT requires 100 - 200 IgG or C3 molecules on the cell for positive reaction
• Ratio of Serum to cells
• reaction medium
• temp
• incubation time
• washing of RBCs
• saline for washing
• addition of AHG
• centrifugation

Ratio of Serum to Cells

• Sensitivity increases with increase in serum to cells: standard is 40:1 ( 2 drops serum, 1 drop 5% v/v suspension of cells
• Weak ab detection is accomplished thru increase in ration, ex: 4 drops serum with 1 drop 3% cell suspension (133;1)

Reaction Medium

• Include albumin, LISS, and polyethylene glycol.
• Albumin: macromolecules allows aggregation from antibody-coated cells: use does provide not advantage over LISS
• Low ionic strength solutions (LISS): enhances antibody uptake w shorter incubation times
• Polyethylene glycol (PEG): concentrate antibody by removing water; Anti-IgH is AHG reagent choice

Temperature

• The reaction rate of the majority of IgG is at 37 degrees C, which is therefore incubation for IAT, while this also is true for complement activation.

Incubation time

• For cells suspended in saline = Vary between 30-130min
• LISS or PEG = May be shortened to 10-15min
• Extended Incubation in the LISS technique has caused Ab to illude from the RBCs ( Decrease the Sensitivity of the test )

Washing of RBCs

• Required amount of times before adding AHG in both DAT and IAT is x3, removes any serum globulins; inadequate washing= false - results
• Always performed /w minimal downtime, can be controlled w check cells or group O
• All solutions should be discarded after use /w each cell washing to limit the possibility of test dilution

Saline for Washing

• PH washing range = 7.2 - 7.4
• Stored/ old solutions have been show to lower PH + accelerate ab ellution = - results
• PH changes = implications when using Monoclonal AHG /w their reactive ranges
• Addition of AHG: Should take place + cells to minimize change of ab eluding form the cells, vol used, or by the manufacture
• Voak= Adding 2 volumes of AHG counteracting washing problems where low serum levels exist, ab neutralization is only a concern in left serum and   Residual serum with serum to neutralize the antiC33 and antiC3d

Centrifugation for Reading

•  Is vital, cell pellet hemagglutination after as well
• BBER recommendations, AHG use RCF’s (1,ooo)for 20 sec
• Chapter = 500 RCF’s for ~15sec

Sources of Error

• See: 5-1for + and - reasons in Anti-Error during reaction
• EDTA must be using to collect blood samples for Dat’s in order to prevent the in vitro

Modified and Automated Antiglobulin Test Techniques

• Low Ionic Polybrene Technique
• cells for lip rapidly sensitize + antibody
• rouleaux-forming polybrene reagent for a potency sensitized cells approach
• ionic solution, high, reverses the rouleaux Enzyme linked (ELAT): RBC suspension added to a microtiter and lysed by an enzyme

Solid Phase Technology

• Can test both directions + phases. Is where a fixated is tested/w an agent that can then be recognized. Available readers = automation

The Gel Test