Presentation on theme: "When can you use an antibody to find another antibody?"— Presentation transcript:
1When can you use an antibody to find another antibody?
2Basic Principle, Procedures, and Applications The Antiglobulin TestBasic Principle, Procedures, and Applications
3The Acquired Immune Response From our Immunology Review we know:The body makes antibodies in response to foreign antigen.These antibodies coat the foreign object leading to:Clearance of the foreign antigen by the RESLysis of the foreign object via complement activationIgG is the predominant antibody produced in most responsesIncomplete antibodyUsually not detected at room temperature/immediate spin phase of testing
4The Antiglobulin Test Y Y Y The purpose of the antiglobulin test is to detect cells that have become coated with antibodies &/or complement.The test is also known as the Coombs test.YYYThe antiglobulin test was first described in 1908, but wasn’t applied to human testing until 1945 by Coombs, Mourant, and Race. Prior to this, only directly agglutinating (IgM) antibodies could be detected. The antiglobulin test (AGT) soon lead to the detection and definition of many previously unrecognized red cell antigen systems, the first of which was the Kell system.
5Anti-Human GlobulinThe main reagent used in the antiglobulin test is anti-human globulin (AHG).Also called Coombs serum.Anti-human globulin (AHG) is an IgG antibody directed against human immunoglobulins or complement components.
6AHG ProductionTraditionally, anti-human globulin was produced by injecting human globulins into rabbits. The rabbits produced antibodies to the human globulins (anti-human globulin). These antibodies were harvested and purified. They are the active ingredient in the AHG reagent. This is an example of a heterophile or xenophile antibody.Today, monoclonal techniques are used to produce most of the AHG reagent available.
7AHG Production The globulins that AHG may be directed against include: IgGIgMIgAC3
8AHG ContentsPolyspecific AHG reagent contains antibodies to both IgG and C3.Monospecific AHG contains antibodies to either IgG or C3.In other words, polyspecific Coombs serum contains anti-IgG and anti-C3 from a non-human source. Polyspecific AHG is also called polyvalent or broad spectrum AHG.Anti-IgM and anti-IgA AHG are also available, but not routinely used.
9AHG ActionAHG combines with the Fc portion of a sensitizing antibody.This completes the antigen-antibody bridge, allowing agglutination to occur.YYYThe specificity of the antibodies in the AHG reagent are to either the Fc portion of an immunoglobulin molecule (usually IgG) or C3.
10When used to detect clinically significant antibodies, AHG reagent MUST contain anti-IgG. Both monospecific anti-IgG and polyspecific AHG reagent would be acceptable for this purpose.
11YOU MUST ADD COOMBS CONTROL CELLS AND GET POSITIVE RESULTS!!! ANYTIME YOU ARE USING AHG REAGENT, AND GET A NEGATIVE TEST RESULT (by tube)---YOU MUST ADDCOOMBS CONTROL CELLSAND GET POSITIVE RESULTS!!!A negative test with Coombs Control cells invalidates the antiglobulin test. The test would need to be completely repeated.
12Coombs Control Cells Y Y Y Y Y Y Y Rh positive cells coated with anti-D antibodies or cells coated with the C3 portion of complement.Coombs Control Cells will react with the antibody in the AHG reagent.YDYDDYYYYDDDY
13Coombs Control Cells will prove that… Coombs reagent was added.Coombs reagent was active.The wash step was adequate to remove any unbound globulins.The most common error made when performing the antiglobulin test is inadequate washing.Many labs use AHG reagent that has been tinted green to help assure the tech that AHG reagent has been added.The most common error made when performing the antiglobulin test is inadequate washing. Any unbound antibodies may neutralize the AHG reagent once it has been added to the test tube. As few as 1:4000 unbound globulins can neutralize AHG.
14Procedures Direct Antiglobulin Test (DAT) Detects antibody (or complement) sensitizing red cellsIn vivo sensitizationUses patient’s cellsAKA Direct Coombs Test.Red cells that became coated with antibodies in the body are detected by the DAT.
15Procedures Indirect Antiglobulin Test (IAT) Antibody is freeUses incubation at 37oC to force red cell sensitization in vitro.May be used to detect antigens or antibodies.Both DAT and IAT utilize Anti-Human Globulin reagent (AHG).AKA Indirect Coombs Test.In the IAT procedure, the red cells become coated with antibody during the incubation step of the procedure. The IAT uses the patient’s red cells when looking for antigens on those cells. Reagent red cells are used when looking for antibodies in the patient’s plasma.
17Steps to the DAT Procedure (tube method) 1. One drop of patient’s red cells are washed with 0.9% NaCl a minimum of 3 times to remove plasma that may contain unbound antibodies.2. AHG reagent is added.3. Tube is centrifuged.4. If IgG or C3 is coating the cells, agglutination will occur (positive test).This will depend on the type of AHG reagent used i.e. if C3 is coating the cell, and monospecific anti-IgG AHG reagent is used, there will be NO agglutination.If neither is present there will be no agglutination (negative test).5. Each negative test is validated (controlled) through the addition of Coombs Control Cells (also called check cells).Patient’s red cells are at a 2-5% cell suspension in saline.
20What causes a cell to become coated with antibodies in vivo? Hemolytic Transfusion ReactionHemolytic Disease of the Fetus and NewbornDrugsDiseaseWhen investigating a positive DAT, it is important to obtain a thorough history of the patient’s transfusions, transplants, pregnancies, medications and diagnosis.
22Steps to the IAT Procedure (tube method) One or two drops of serum (may contain an antibody) are added to a test tube.One drop of red cells (antigen source) is added to the tube.The tube is incubated at 37oC. The length of incubation is dependant on the medium.Following incubation, the cells are washed with saline a minimum of 3 times, to remove any unbound antibody.Following the final wash, two drops of AHG reagent are added to the dry cell button. The tube is centrifuged and results are read. The tube may be read microscopically, depending on the test medium.Coombs control cells are added to each negative test. The tubes are centrifuged and results read.Red cells are at a 2-5% cell suspension in saline.
24Indirect Antiglobulin Test Antibodies sensitize the red cells during incubation at 37oC.Following the wash step, AHG reagent is added.AHG reagent completes the “bridge” between red cells, allowing for visible agglutination.
26Indirect Antiglobulin Test Looking for in vitro cell sensitization.Uses incubation at 37oC to allow antibody to sensitize red cell.Uses AHG reagent to complete the “bridging” between red cells.Visible agglutination as a positive endpoint.Enhancement reagents may be added during incubation phase to increase sensitization and agglutination.
27Applications Using Patient’s Serum Antibody screenDetects antibodies in patient’s serumUses reagent red cells as a source of known antigenAntibody panelIdentifies antibodies
28Applications Using Patient’s Serum Antiglobulin crossmatchDetermines patient’s compatibility with donorUses donor red cells (antigens) and patient’s serum (antibodies)Usually performed only when a patient has an antibody or a history of antibodiesThe antiglobulin crossmatch (AGXM) gives added assurance that the antibodies in the patient’s serum will not react with antigens on the donor red cells.
29Applications Using Patient’s Cells Antigen TypingWeak D testBoth use commercial anti-serum which contains antibodies, versus the patient’s cells (antigen).Auto control – Patient’s plasma vs. patient’s cellsNOTE:If the patient has a positive DAT, the results of any IAT using the patient’s cells will be invalid.Cells are already coated with antibody before the incubation step!
30Factors affecting the IAT Serum/Cell ratioIncubation temperaturepHLength of incubationTest environment (enhancement media)Serum/cell ratio is most often 2 drops serum to 1 drop cells. Too weak of a cell suspension (too few antigens) = prozone. Too heavy of a cell suspension (too many antigens) = postzoneTemperature 36-38oC (Needs to be at “body temperature” to induce sensitization)Neutral pH (6.5 to 7.5)Incubation time will depend on test environment. Incubate for too short or too long of time for the medium being used and agglutinates will either not have formed or will have formed and be falling apart. Saline environment min inc. Potentiators may be added to decrease the incubation time (anywhere from 10 – 30 minutes).
31POTENTIATORSSome incomplete antibodies will not react in a saline environment.Potentiators are reagents that adjust the test environment.Reduce the zeta potentialPromote agglutinationEnhance antibody uptakeAlso called enhancement media.
3222% AlbuminPolyethylene glycol (PEG) : a low ionic strength medium. Removes water from the test system, thereby concentrating any antibody present.LISSEnzymes
33Sources of Error in the Antiglobulin Test Adequate washCentrifugationProblems with reagents/salineProblems reading reactionsWash – a minimum of 3 times, to remove unbound antibodies that could neutralize AHG reagent. Most common error in the AGT is inadequate wash.Centrifugation- throws cells together in closer contact, allowing agglutination to occur more readily. Too slow or too short of time, not enough contact. Too fast or too long of time, mechanical packing of cells (not agglutination due to antigen-antibody interaction)Reagents/saline – Neutralization, bacterial contaminationReading reactions – Debris often confused with microscopic positive reactions. If complement is present, may see hemolysis (positive reaction)