The Antiglobulin Test
Polyspecific AHG reagents contain: A.anti-IgG B.anti-C3d C.anti-IgG and anti-IgM D.anti-IgG and anti-C3d
D.anti-IgG and anti-C3d Polyspecific AHG reagents must contain anti-IgG and anti-C3d.
In which of the following is the indirect antiglobulin test utilized? A.reverse ABO testing B.immediate spin crossmatch C.C antigen testing D.antibody detection (screening) test
D.antibody detection (screening) test Following incubation at 37°C, antibody screening (detection) tests must include an indirect antiglobulin phase to facilitate detection of clinically significant antibodies.
In the direct antiglobulin test, the antiglobulin reagent is used to: A.mediate hemolysis of indicator red blood cells by providing complement B.precipitate anti-erythrocyte antibodies C.measure antibodies in a test serum by fixing complement D.detect preexisting antibodies on erythrocytes
D.detect preexisting antibodies on erythrocytes Antiglobulin reagent is used to detect the presence of red cells, coated in vivo with IgG and/or C3d. Antiglobulin reagent may be polyspecific (contains an anti-IgG and anti-C3d) or monospecific (anti-IgG or anti-C3d).
Monoclonal blood banking reagents have which of the following as a disadvantage? A.little to no batch variation B.cost effectiveness C.high antigen efficiency D.overspecificity
D.overspecificity Overspecificity can be a disadvantage of monoclonal reagents. Blending these reagents with other monoclonal reagents or polyclonal reagents has been useful in overcoming this disadvantage.
The immune response to red cell antigens with numerous epitopes results in a heterogeneous population of antibodies referred to as: A.bivalent B.epitope-specific C.monoclonal D.polyclonal
D.polyclonal These antibodies are referred to as polyclonal.
Which of the following might cause a false-negative indirect antiglobulin test (IAT)? A.over-reading B.IgG-coated screening cells C.addition of an extra drop of serum D.too heavy a cell suspension
D.too heavy a cell suspension Weak antibodies may be missed if there are excess RBC antigens as there may be too few antibodies to bind to red cell antigens.
Direct Antiglobulin Tests (DAT) vs Indirect Antiglobulin Test (IAT)
DAT detects in vivo RBC sensitization; detects Abs or complement on surface of RBCs IAT detects in vitro RBC sensitization in test tube; detects Abs in the serum
Why would a DAT be needed?
to detect IgG or complement proteins bound to patient cells which is a consequence of certain clinical events, including autoimmune hemolytic anemia, HDFN, a drug-related mechanism, or an antibody reaction to transfused red cells. A positive DAT is an important indicator of potential immune-mediated red cell destruction in the body.
Why would an IAT be needed?
to detect in vitro sensitization of red cells. A positive IAT indicates a specific reaction between an antibody in the serum/plasma and an antigen present on the red cells. -used in several Blood Bank procedures to enhance /detect Ab/Ag reaction a. Detection of unexpected Abs b. Crossmatching c. Detecting RBC Ags not demonstrated by other methods
What are sources of error in an IAT?
• Inadequate washing of cells = false negative • Contamination of AHG = false negative • Delay in steps = false negative • Omission of AHG reagent = false negative • Undercentrifugation = false negative • Cells with Positive DAT can give a false Positive IAT • Contaminated glassware = false positive • Overcentrifugation = false positive
DAT procedure
A - patient red cells are washed to remove unbound immunoglobulins. B - AHG reagent, either polyspecific or monospecific, is added If either IgG or C3d molecules are present on the patient's red cells, agglutination is observed. The patient has a positive DAT result.
Which of the following tests is most commonly used to detect antibodies attached to a patient's red blood cells in vivo? A.direct antiglobulin B.complement fixation C.indirect antiglobulin D.immunofluorescence
A.direct antiglobulin The direct antiglobulin test (DAT) is used to identify red blood cells that have been coated with antibody in vivo.
A false-negative direct antiglobulin test can be the result of: A.neutralized AHG reagent B.sample from gel-separator tubes C.overcentrifugation D.dirty glassware
A.neutralized AHG reagent Neutralized AHG reagent, no longer able to bind to IgG immunoglobulin, can result in a false-negative test result.
Polyspecific AHG
Ab can recognize >1 Ag -contains both anti-IgG and anti-C3d antibodies and detects both IgG and C3d molecules on red cells -if the test result is positive, the test is repeated using monospecific AHG regents Anti-IgG and Anti-complement
Monospecific AHG
Ab can recognize only one Ag -specific for IgG and complement separately to determine which molecule was on the red cell. No agglutination after the addition of polyspecific AHG reagent is interpreted as a negative DAT Anti-IgG or Anti-complement
Polyclonal
Ab originated from >1 parent cell mixture of antibodies that may be directed at different epitopes of the same antigen Mixture of IgM and IgG
Monoclonal
Ab originated from one parent cell unique specificity for a particular epitope either IgG or IgM
Polyspecific reagents used in the direct antiglobulin test should have specificity for: A.IgG and IgA B.IgG and C3d C.IgM and IgA D.IgM and C3d
B.IgG and C3d Polyspecific AHG contains anti-IgG and anti-C3d.
The direct antiglobulin test in a suspected case of warm autoimmune hemolytic anemia is positive. Which of the following monospecific reagents would be used in further direct antiglobulin testing? A.anti-C3b B.anti-C3d C.anti-C4 D.anti-IgM
B.anti-C3d Anti-human globulin reagents used to evaluate suspected cases of autoimmune hemolytic anemia must contain anti-C3d and anti-IgG. Once a positive direct antiglobulin is noted using a polyspecific reagent, testing with monospecific anti-C3d and monospecific anti-IgG would be done.
In which of the following clinical situations will the direct antiglobulin test be positive? A.sickle cell disease B.hemolytic disease of the fetus and newborn C.posttransfusion purpura D.multiple myeloma
B.hemolytic disease of the fetus and newborn Hemolytic disease of the fetus and newborn will demonstrate a positive direct antiglobulin test.
Review the following schematic diagram: PATIENT SERUM + REAGENT GROUP "O" CELLS INCUBATE -> READ FOR AGGLUTINATION WASH ->ADD AHG -> AGGLUTINATION OBSERVED The next step would be to: A.add "check cells" as a confirmatory measure B.identify the cause of the agglutination C.perform an elution technique D.perform a direct antiglobulin test
B.identify the cause of the agglutination Agglutination at AHG phase indicates the presence of clinically significant antibody, indicating the need for antibody identification.
A group B, Rh-negative patient has a positive DAT. Which of the following situations would occur? A.all major crossmatches would be incompatible B.the weak D test and control would be positive C.the antibody screening test would be positive D.the forward and reverse ABO groupings would not agree
B.the weak D test and control would be positive A positive DAT will interfere with weak D testing causing both the patient and control to demonstrate positive results. Any positive result in the control tube invalidates any results.
The addition of antibody-sensitized red cells (Check Cells) to all negative anti-human globulin (AHG) tests ensures that: A.the test was interpreted correctly B.the test was incubated at the correct temperature C.AHG reagent was added to each test D.patient serum was added to each test
C.AHG reagent was added to each test Failure to demonstrate hemagglutination following the addition of IgG-sensitized red blood cells (Check Cells) suggests the possibility of inadequate washing to remove unbound globulin, failure to add AHG reagent, or faulty/nonreactive AHG.
AHG (Coombs) control cells: A.can be used as a positive control for anti-C3 reagents B.can be used only for the indirect antiglobulin test C.are coated only with IgG antibody D.must be used to confirm all positive antiglobulin reactions
C.are coated only with IgG antibody AHG control cells are IgG-sensitized cells that react with the anti-IgG in the AHG reagent to demonstrate AHG was added and not neutralized by insufficient washing of the tests prior to its addition.
A false-positive indirect antiglobulin test can be the result of: A.insufficient saline washing of red cells B.inadequate incubation time C.overcentrifugation D.dissociation of cell bound IgG
C.overcentrifugation Overcentrifugation of the test can result in a false-positive result.
A patient received 2 units of Red Blood Cells and had a delayed transfusion reaction. Pretransfusion antibody screening records indicate no agglutination except after the addition of IgG-sensitized cells. Repeat testing of the pretransfusion specimen detected an antibody at the antiglobulin phase. What is the most likely explanation for the original results? A.red cells were overwashed B.centrifugation time was prolonged C.patient's serum was omitted from the original testing D.antiglobulin reagent was neutralized
C.patient's serum was omitted from the original testing Initial result was most likely a false-negative result due to the omission of patient serum. This would explain the initial negative result followed by the subsequent positive result.
Crossmatch results at the antiglobulin phase were negative. When 1 drop of check cells was added, no agglutination was seen. The most likely explanation is that the: A.red cells were overwashed B.centrifuge speed was set too high C.residual patient serum inactivated the AHG reagent D.laboratorian did not add enough check cells
C.residual patient serum inactivated the AHG reagent A negative reaction after the addition of check cells indicates AHG serum was not present. Inadequate washing of red cells may leave residual patient serum behind, which can neutralize AHG serum.
How to detect potential neutralization?
IgG-sensitized cells are added to tubes with negative reactions. After centrifugation, a positive reaction should be observed to confirm that washing was adequate.
IAT procedure
In the IAT, a source of antibody and red cells are incubated at 37° C for a specified time to allow Ag-Ab reactions to occur. After incubation, the red cells are washed to remove unbound molecules. AHG reagent, monospecific anti-IgG is added. If IgG is present on the red cells, agglutination is observed. The result is a positive IAT.
Why is it important to wash RBCs for an antiglobulin test?
It is essential that the red cells be washed with saline to remove any unbound molecules before the addition of the AHG reagent. If the test red cells are inadequately washed, any unbound antibody or complement present in the test can potentially bind to the AHG reagent and inhibit its reaction with antibody or complement molecules attached to the red cells. This is neutralization of the AHG. It's a source of error that can mask a positive antiglobulin test by blocking antibody sites, causing a negative reaction.
Can we use a clotted sample?
No. Because complement can attach nonspecifically to red cells when samples are stored, it is important to use an anticoagulated EDTA sample when performing this test. Because the EDTA negates the in vitro activation of the complement pathway, the test detects only complement proteins that have been bound to the red cells in vivo.
RBC washing procedure
The washing step of an antiglobulin test requires the filling of test tubes with saline to mix with the red cells already present in the tube. The saline-suspended red cells are centrifuged. The saline wash is decanted, and this process is repeated for two or three cycles. On completion of the last wash, the saline is removed, and the tube is blotted dry to remove most traces of saline.
Principle of antiglobulin test
Uses a reagent that has been prepared by injecting rabbits with human antibody molecules (human IgG) and complement proteins. The injected proteins are recognized as foreign antigens, stimulating the animal's immune system to produce antibodies to human antibody molecules and complement proteins. The reagent, polyspecific AHG, contains antibodies to IgG molecules (anti-IgG) and complement proteins (anti-C3d, anti-C3b). This AHG reagent reacts with human IgG antibody and complement proteins. If the red cells are sensitized with IgG or complement, the AHG reagent crosslinkes the sensitized cells and causes agglutination. The formation of agglutinated red cells after the addition of AHG shows that IgG or complement proteins were attached to the red cells - this is a positive antiglobulin test.
What factors can influence an IAT?
a. temperature • 370C used - IgG binds best • Do not use only RT because it only detects clinically insignificant Abs • RT ok for DAT because detecting Ab already attached to RBCs b. medium • saline - attachment is slow • albumin - attachment is enhanced • LISS - attachment is enhanced and shorter incubation time c. ratio cells/serum • 2 drops serum + 1 drop 5% suspension of cells • can increase serum to 5-10 drops to enhance reaction but check protocol d. incubation time • 15-30 mins minimum time • LISS = 10 minutes
antiglobulin test
detects IgG molecules and complement protein molecules that have attached (sensitized) to red cells but have not resulted in a visible agglutination reaction