MODIFIED AND AUTOMATED ANTIGLOBULIN TEST TECHNIQUES CONT. Flashcards
first described by Lalezari and Jiang in 1980
Low Ionic Polybrene (LIP) technique
offers a manual adaptation of an automated method for sensitizing cells with antibodies.
Low Ionic Polybrene (LIP) technique
Principle: relies on low ionic conditions to facilitate rapid sensitization of cells with antibodies.
LIP technique
Polybrene, a compound known for forming rouleaux (clumps of red blood cells), is added to aid in bringing the sensitized cells close together, allowing for cross-linking by the attached antibodies.
LIP technique
Procedure:
1.Initially, low ionic conditions are created to sensitize the cells with antibodies quickly. Polybrene is added to facilitate the formation of rouleaux.
2.Following this, a high ionic strength solution is introduced to reverse the rouleaux formation. If agglutination persists, it indicates the presence of antibody binding to the red blood cells.
3.If necessary, the test can be further carried out using an antiglobulin (AHG) technique. In this case, a monospecific anti-IgG reagent is required due to the presence of considerable amounts of C4 and C3 coating the cells under low ionic conditions, which could lead to false positive reactions if a polyspecific reagent were used.
LIP technique
is increasingly used in blood group serology due to its convenience and efficiency.
Microplate Adaptation
Redman and colleagues adapted the LIP technique for use in microplates.
Microplate Adaptation
While their report didn’t include the use of an AHG phase, it can be easily incorporated into the process.
Microplate Adaptation
offers a relatively quick and straightforward method for detecting antibody binding to red blood cells.
LIP technique
Its adaptation for use in microplates adds further convenience, making it suitable for high-throughput applications in blood group serology laboratories.
LIP technique
it’s crucial to ensure the appropriate choice of reagents, including the use of monospecific anti-IgG reagents if proceeding to an AHG phase, to avoid false-
positive reactions
LIP technique
provides a manual approach for sensitizing red blood cells with antibodies under low ionic conditions, with potential adaptation for use in microplate technology, offering convenience and efficiency in blood group serology testing.
LIP technique
is a variation of the antiglobulin test (also known as the Coombs test) that employs an enzyme-labeled antiglobulin reagent
Enzyme-Linked Antiglobulin Test
: Initially, a suspension of red blood cells (RBCs) from the patient’s sample is prepared.
1.Preparation of RBC Suspension
This suspension typically contains RBCs that may have been sensitized with antibodies, which could indicate various immune-related conditions such as autoimmune hemolytic anemia or hemolytic disease of the newborn.
1.Preparation of RBC Suspension
: Microtiter wells are used as the reaction vessels for this test.
- Microtiter Plate Setup
A small volume of the RBC suspension is added to each well of the microtiter plate.
- Microtiter Plate Setup
: The RBCs in the microtiter wells are washed with saline to remove any unbound or non-specifically bound components, ensuring that only specific antibody-RBC complexes remain.
- Washing Step
: An antiglobulin reagent that has been labeled with an enzyme (such as horseradish peroxidase or alkaline phosphatase) is added to the wells.
- Addition of Enzyme-Labeled AHG
This will bind specifically to any IgG antibodies that have sensitized the RBCs present in the wells.
- Addition of Enzyme-Labeled AHG
: The microtiter plate is then incubated to allow sufficient time for the enzyme-labeled antiglobulin to bind to any IgG-sensitized RBCs present in the wells.
- Incubation and Binding
: After the incubation period, any excess enzyme-labeled antiglobulin reagent is washed away to minimize background noise and non-specific binding.
- Removal of Excess Reagent
: A substrate specific to the enzyme used for labeling the AHG is added to each well.
- Addition of Enzyme Substrate
When the enzyme reacts with the substrate, it produces a color change or a fluorescent signal.
- Addition of Enzyme Substrate
: The amount of color or fluorescence produced in each well is measured spectrophotometrically or using a specialized plate reader.
- Colorimetric Measurement
The optical density is usually measured at a specific wavelength, commonly 405 nm for many enzyme substrates.
- Colorimetric Measurement
: The intensity of the color or fluorescence signal is proportional to the amount of antibody present in the sample.
- Quantification of Antibody
This allows for the quantitative determination of the level of IgG antibodies bound to the RBCs.
- Quantification of Antibody
: From the measured optical density or fluorescence intensity, the number of IgG molecules per RBC can be estimated, providing valuable information about the antibody load and the severity of the immune reaction
- Determination of IgG Molecules per RBC
offers a sensitive and quantitative method for detecting and quantifying IgG antibodies bound to red blood cells, making it a valuable tool in the diagnosis and monitoring of various immune mediated hemolytic disorders.
Enzyme-Linked Antiglobulin Test
has revolutionized the performance of antiglobulin tests, offering increased sensitivity, specificity, and automation potential.
Solid-phase technology
: This approach involves the immobilization of antigens or antibodies on a solid surface, typically microplate wells or test tubes.
1.Solid-Phase Methodology
The interaction between the immobilized antigen or antibody and the corresponding antibody or antigen in the test sample is then detected.
1.Solid-Phase Methodology
: In the direct antiglobulin test performed using solid-phase methodology, antibodies specific for red blood cell (RBC) antigens are attached to the surface of microplate wells.
- Direct Antiglobulin Test (DAT)
Patient RBCs are then added to the wells. If the patient’s RBCs carry the corresponding antigens recognized by the immobilized antibodies, they will bind to the well’s surface, resulting in a positive reaction.
- Direct Antiglobulin Test (DAT)
This method allows for the direct detection of antibodies bound to the patient’s RBCs.
- Direct Antiglobulin Test (DAT)
: In the [?] using solid-phase methodology, known RBCs with specific antigens are bound to the surface of the microplate wells, typically treated with agents like glutaraldehyde or poly L lysine for fixation.
- Indirect Antiglobulin Test (IAT)
Patient serum containing antibodies is then added to the wells.
- Indirect Antiglobulin Test (IAT)
This method enables the detection of antibodies present in the patient’s serum.
- Indirect Antiglobulin Test (IAT)
If the patient serum contains antibodies specific to the antigens present on the fixed RBCs, a positive reaction occurs.
- Indirect Antiglobulin Test (IAT)
: Solid-phase technology can be easily adapted for semi-automation or full automation using microplate readers.
- Automation Potential
Automated systems offer advantages such as increased throughput, reduced hands-on time, and improved standardization of results.
- Automation Potential
: Companies like Immucor Incorporated offer solid-phase systems specifically designed for the detection and identification of alloantibodies.
- Commercial Systems
These systems utilize polystryrene microtitration strip wells coated with group O reagent RBC membranes.
- Commercial Systems
Patient or donor IgG antibodies are bound to the membrane antigens.
- Commercial Systems
Following incubation and washing steps, a suspension of anti-IgG coated indicator RBCs is added.
- Commercial Systems
Positive reactions result in the adherence of indicator RBCs, forming antiIgG-IgG complexes and a second immobilized RBC laye
- Commercial Systems
also known as gel column agglutination
Gel Test
is a highly effective method for detecting red blood cell (RBC) antigen-antibody reactions
Gel Test
Gel Test The gel test utilizes a chamber filled with a polyacrylamide gel. This gel acts as a matrix or trap, allowing unagglutinated RBCs to form pellets at the bottom of the tube while trapping agglutinated RBCs within the gel matrix.
Gel Test
As a result, negative reactions appear as pellets at the bottom of the microtube, while positive reactions are fixed within the gel.
Gel Test
- Types of Gel Tests:
- Neutral Gel Test
- Specific Gel Test
: This type of gel does not contain any specific reagent and relies solely on its property of trapping agglutinates.
- Neutral Gel Test
It is commonly used for antibody screening and identification, enzymetreated or untreated RBCs, and reverse ABO typing.
- Neutral Gel Test
: Specific reagents, such as monoclonal antibodies or antigens, are incorporated into the gel.
- Specific Gel Test
This type of gel test is useful for determining specific antigens on the RBC surface.
- Specific Gel Test
is a valuable application of the gel test, suitable for both the indirect antiglobulin test (IAT) and the direct antiglobulin test (DAT)
Low Ionic Antiglobulin Test (GLIAT)
antiglobulin reagent (AHG) is incorporated into the gel.
Low Ionic Antiglobulin Test (GLIAT)
For example, in an IAT gel test, a small amount of RBC suspension is pipetted onto a gel containing AHG.
Low Ionic Antiglobulin Test (GLIAT)
After incubation and centrifugation, positive and negative reactions are separated within the gel, providing a safe, reliable, and easy-to-read AHG test
Low Ionic Antiglobulin Test (GLIAT)
Low Ionic Antiglobulin Test (GLIAT) Procedure:
a. For the [?], a small volume of RBC suspension in low ionicstrength solution (LISS) is added to the top of each microtube of the gel card designed for Coombs testing.
b. The cards are then [?] to allow the reaction to occur.
c. In the case of a positive reaction, [?] such as anti-IgG or anti-C3d can be used in the gel test to determine the specificity of the antibody.
DAT
centrifuged
monospecific reagents
The gel test offers several advantages, including ease of use, rapid results, and the ability to perform multiple tests simultaneously.
Gel Test
It provides a clear separation between positive and negative reactions, making interpretation straightforward.
Gel Test
The gel format minimizes the need for a washing phase, reducing hands-on time and potential sources of error.
Gel Test
The comparison between the tube and gel techniques in performing Direct Antiglobulin Tests (DATs) has been the subject of several studies, highlighting differences in sensitivity and specificity between the two methods.
Traditional Tube Technique Versus the Gel Test in the DAT
: This study compared the tube and gel tests in evaluating ABO Hemolytic Disease of the Newborn (HDN).
1.Chuansumrit et al.’s Study
Study The gel test showed higher sensitivity in detecting positive DAT results in infants with ABO incompatibility compared to the tube test.
1.Chuansumrit et al.’s
This higher sensitivity of the gel test, particularly in detecting IgG antibodies associated with ABO HDN, suggests its superiority in this clinical scenario.
1.Chuansumrit et al.’s Study
: This report described a case of Autoimmune Hemolytic Anemia (AIHA) where the gel test yielded a positive result while the traditional tube test was negative.
- Lai et al.’s Case Report
The gel test’s no-wash nature prevented the loss of low-affinity autoantibodies during washing, potentially explaining the discrepancy between the two methods.
- Lai et al.’s Case Report
This case underscores the importance of considering the no-wash aspect of the gel test in situations where low-affinity antibodies may be present.
- Lai et al.’s Case Report
: This study compared the gel test with the tube and enzyme-linked antiglobulin test (ELAT) techniques.
- Mitek et al.’s Comparative Study
It found the gel test to be more sensitive, particularly in hypergammaglobulinemic patients, where it detected positive results due to IgG antibodies that were missed by the tube and ELAT methods.
- Mitek et al.’s Comparative Study
These findings highlight the potential advantage of the gel test in detecting antibodies in certain patient populations.
- Mitek et al.’s Comparative Study
: The differences between the tube and gel techniques in DATs underscore the importance for blood banks to be aware of the methodological variations and their impact on test results.
- Implications for Blood Banks
While the gel test offers advantages such as ease of use and rapid results, it may yield different results compared to the tube technique, particularly in detecting certain types of antibodies.
- Implications for Blood Banks
: With ongoing advancements in blood bank technology and evolving priorities in antibody screening and crossmatching, the role of the antiglobulin test may continue to evolve.
- Future Directions
Additional comparative studies will be valuable in further understanding the strengths and limitations of different testing methodologies.
- Future Directions
While the antiglobulin test remains a cornerstone in blood bank testing for detecting clinically significant antibodies and immune hemolysis, the choice between the tube and gel techniques should be made considering the specific clinical context and the potential impact on test sensitivity and specificity.
- Future Directions
