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Questions and Answers
A patient's blood sample yields a positive DAT result. What is the MOST appropriate next step to identify the cause of the sensitization?
A patient's blood sample yields a positive DAT result. What is the MOST appropriate next step to identify the cause of the sensitization?
- Order a GLIAT to determine the antibody specificity.
- Immediately begin elution studies to remove the antibody from the red cells.
- Perform an IAT to confirm the presence of in-vitro sensitization.
- Use a DAT panel with monospecific anti-IgG and anti-C3d to determine the specific type of protein sensitizing the RBC. (correct)
In which scenario is the Indirect Antiglobulin Test (IAT) LEAST likely to be utilized?
In which scenario is the Indirect Antiglobulin Test (IAT) LEAST likely to be utilized?
- Determining the presence of a specific antigen on a patient's red blood cells.
- Performing compatibility testing before a blood transfusion.
- Investigating a suspected case of Hemolytic Disease of the Newborn (HDN). (correct)
- Screening a patient's serum for unexpected antibodies.
Why are AHG reagents containing anti-IgG necessary for detecting IgG antibodies in the antiglobulin test?
Why are AHG reagents containing anti-IgG necessary for detecting IgG antibodies in the antiglobulin test?
- The IgG monomeric structure is too small to directly agglutinate sensitized RBCs. (correct)
- IgG antibodies directly agglutinate sensitized RBCs, but AHG reagents enhance the reaction for easier visualization.
- AHG reagents neutralize interfering substances that inhibit IgG antibody binding.
- AHG reagents convert IgM antibodies into IgG antibodies to facilitate detection.
A technologist observes complement activation during a Direct Antiglobulin Test (DAT). Which preanalytical error is the MOST likely cause?
A technologist observes complement activation during a Direct Antiglobulin Test (DAT). Which preanalytical error is the MOST likely cause?
What is a key advantage of using Gel technology in Low Ionic Antiglobulin Test (GLIAT) compared to traditional tube methods?
What is a key advantage of using Gel technology in Low Ionic Antiglobulin Test (GLIAT) compared to traditional tube methods?
What is the primary difference between polyclonal and monoclonal AHG production?
What is the primary difference between polyclonal and monoclonal AHG production?
Why might a laboratory choose to use a blend of monoclonal anti-C3b and anti-C3d with polyclonal anti-IgG in an AHG reagent?
Why might a laboratory choose to use a blend of monoclonal anti-C3b and anti-C3d with polyclonal anti-IgG in an AHG reagent?
In the context of the Indirect Antiglobulin Test (IAT), what is a key limitation of using monospecific anti-IgG AHG compared to polyspecific AHG?
In the context of the Indirect Antiglobulin Test (IAT), what is a key limitation of using monospecific anti-IgG AHG compared to polyspecific AHG?
When evaluating a weakly positive AHG reaction after performing an IAT, what is the initial recommended step?
When evaluating a weakly positive AHG reaction after performing an IAT, what is the initial recommended step?
Which of the following is an application of the Direct Antiglobulin Test (DAT)?
Which of the following is an application of the Direct Antiglobulin Test (DAT)?
A patient has a positive DAT. Which of the following questions is MOST relevant to determining the cause of the positive DAT?
A patient has a positive DAT. Which of the following questions is MOST relevant to determining the cause of the positive DAT?
In investigating a positive DAT result, what is the significance of determining whether there is evidence of in vivo hemolysis?
In investigating a positive DAT result, what is the significance of determining whether there is evidence of in vivo hemolysis?
Which factor, when altered in the Antiglobulin Test (AGT), would most likely lead to a false-positive result due to non-specific aggregation?
Which factor, when altered in the Antiglobulin Test (AGT), would most likely lead to a false-positive result due to non-specific aggregation?
In a compatibility test, if the indirect antiglobulin test (IAT) is performed and the result indicates the presence of an unexpected antibody, what is the next appropriate step?
In a compatibility test, if the indirect antiglobulin test (IAT) is performed and the result indicates the presence of an unexpected antibody, what is the next appropriate step?
How does Polyethylene Glycol (PEG) enhance antibody detection in the Antiglobulin Test (AGT)?
How does Polyethylene Glycol (PEG) enhance antibody detection in the Antiglobulin Test (AGT)?
A technologist observes rouleaux formation after adding polybrene in a Low Ionic Polybrene Technique. What action should be taken to reverse this effect and ensure accurate test results?
A technologist observes rouleaux formation after adding polybrene in a Low Ionic Polybrene Technique. What action should be taken to reverse this effect and ensure accurate test results?
If the temperature during the Antiglobulin Test (AGT) incubation period deviates significantly from 37°C, what is the most likely consequence?
If the temperature during the Antiglobulin Test (AGT) incubation period deviates significantly from 37°C, what is the most likely consequence?
A technologist is performing a gel test using a neutral gel card for antibody screening and observes a solid line of agglutinated red cells at the top of the gel column. What is the most likely interpretation of this result?
A technologist is performing a gel test using a neutral gel card for antibody screening and observes a solid line of agglutinated red cells at the top of the gel column. What is the most likely interpretation of this result?
Which of the following is the primary reason for using IgG monospecific AHG reagent in the Low Ionic Polybrene Technique?
Which of the following is the primary reason for using IgG monospecific AHG reagent in the Low Ionic Polybrene Technique?
In a scenario where a patient has a positive Direct Antiglobulin Test (DAT) and is suspected of having a drug-induced hemolytic anemia, what is the most appropriate follow-up test to confirm this diagnosis?
In a scenario where a patient has a positive Direct Antiglobulin Test (DAT) and is suspected of having a drug-induced hemolytic anemia, what is the most appropriate follow-up test to confirm this diagnosis?
A principle of the antiglobulin test is:
A principle of the antiglobulin test is:
Polyspecific AHG reagent contains:
Polyspecific AHG reagent contains:
Monoclonal anti-C3d is:
Monoclonal anti-C3d is:
Which of the following is a clinically significant antibody whose detection has been reported in some instances to be dependent on anticomplement activity in polyspecific AHG?
Which of the following is a clinically significant antibody whose detection has been reported in some instances to be dependent on anticomplement activity in polyspecific AHG?
After the addition of IgG-coated RBCs (check cells) to a negative AHG reaction during an antibody screen, a negative result is observed. Which of the following is a correct interpretation?
After the addition of IgG-coated RBCs (check cells) to a negative AHG reaction during an antibody screen, a negative result is observed. Which of the following is a correct interpretation?
RBCs must be washed in saline at least three times before the addition of AHG reagent to:
RBCs must be washed in saline at least three times before the addition of AHG reagent to:
An in vitro phenomenon associated with a positive IAT is:
An in vitro phenomenon associated with a positive IAT is:
False-positive DAT results are most often associated with:
False-positive DAT results are most often associated with:
Polyethylene glycol enhances antigen-antibody reactions by:
Polyethylene glycol enhances antigen-antibody reactions by:
Solid-phase antibody screening is based on:
Solid-phase antibody screening is based on:
A positive DAT may be found in which of the following situations?
A positive DAT may be found in which of the following situations?
What do Coombs’ control cells consist of?
What do Coombs’ control cells consist of?
Which of the following methods requires the use of check cells?
Which of the following methods requires the use of check cells?
Which factor can affect AHG testing, yet is uncontrollable in the lab?
Which factor can affect AHG testing, yet is uncontrollable in the lab?
If you had the authority to decide which primary AHG methodology to utilize at your lab, which method would you choose based on the knowledge that the majority of the staff are generalists?
If you had the authority to decide which primary AHG methodology to utilize at your lab, which method would you choose based on the knowledge that the majority of the staff are generalists?
A 27-year-old group O mother has just given birth to a beautiful group A baby girl. Since the mother has IgG anti-A in her plasma, it is likely that the baby is experiencing some in vivo red cell destruction. Which of the following methods and tests would be most effective at detecting the anti-A on the baby’s RBCs?
A 27-year-old group O mother has just given birth to a beautiful group A baby girl. Since the mother has IgG anti-A in her plasma, it is likely that the baby is experiencing some in vivo red cell destruction. Which of the following methods and tests would be most effective at detecting the anti-A on the baby’s RBCs?
Flashcards
Polyspecific AHG
Polyspecific AHG
Contains antibodies to human IgG and C3d complement components.
Monospecific AHG
Monospecific AHG
Contains either anti-IgG or anti-C3b/C3d, but not both.
Polyclonal AHG Production
Polyclonal AHG Production
AHG reagents produced by immunizing rabbits, recognizing different antigenic epitopes.
Monoclonal AHG Production
Monoclonal AHG Production
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Direct Antiglobulin Test (DAT)
Direct Antiglobulin Test (DAT)
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Indirect Antiglobulin Test (IAT)
Indirect Antiglobulin Test (IAT)
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DAT Applications
DAT Applications
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Antigen determination
Antigen determination
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GLIAT Use
GLIAT Use
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Antiglobulin test
Antiglobulin test
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Why use AHG?
Why use AHG?
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Why use EDTA for DAT?
Why use EDTA for DAT?
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IAT (Indirect Antiglobulin Test)
IAT (Indirect Antiglobulin Test)
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Serum to Cells Ratio
Serum to Cells Ratio
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Albumin (bovine) in AGT
Albumin (bovine) in AGT
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LISS in AGT
LISS in AGT
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PEG in AGT
PEG in AGT
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AGT Temperature
AGT Temperature
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Washing RBCs in AGT
Washing RBCs in AGT
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Low Ionic Polybrene Technique
Low Ionic Polybrene Technique
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The Gel Test
The Gel Test
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Study Notes
- The antiglobulin test detects RBCs sensitized by IgG alloantibodies, IgG autoantibodies, and/or complement components
- AHG reagents with anti-IgG are needed to detect IgG antibodies since the IgG monomeric structure is too small to directly agglutinate sensitized RBCs
AHG Reagent
- Polyspecific AHG contains antibodies to human IgG and the C3d component of human complement (Anti-C3b, Anti-C4b, and C4d)
- Monospecific AHG contains either anti-IgG or anti-C3b or C3d
- Anti-IgG are antibodies specific for the Fc fragment of the gamma heavy chain of the IgG
- Anti-complement is for anti-C3b or anti-C3d
Preparation of AHG
- Polyclonal AHG reagents are produced by immunizing one colony of rabbits with human immunoglobulin (IgG) antigen and another colony with human C3 antigen
- This reagent can recognize different antigenic epitopes of the same antigen but with different affinities
- Monoclonal antibodies are used to produce AHG with high titer and specificities antibody to IgG and C3
- Monoclonal antibodies recognize a single epitope of antigen, which is a "disadvantage"
- Monoclonal antibodies to complement component may be blended with polyclonal anti-IgG to achieve a potent reagent that gives fewer false-positive reactions as a result of anticomplement
AHG Reagents
- AHG reagent contains IgG, C3, buffer, stabilizer, bacteriostatic agent, and green dye
- Monospecific anti-IgG is usually polyclonal
- Monospecific anticomplement reagents are blend of monoclonal anti-C3b and monoclonal anti-C3d
Use of Polyspecific Versus Monospecific AHG in The IAT
- Some antibodies are undetectable with monospecific anti-IgG but detectable with anticomplement, and polyspecific AHG can give unwanted positive reactions not caused by clinically significant antibodies
- The first step in evaluating is to repeat with prewarm technique, where 60% become negative
- Polyspecific AHG reagent is used for compatibility testing and indirect test
AHG Reagent and the DAT
- DAT detects in vivo sensitization of RBC with IgG and complement and Anti-C3d is used as international reference reagent
- Polyclonal AHG sera are prepared by injecting human globulins into rabbits, and an immune stimulus triggers production of antibody to human serum
- Hybridoma technology is used to produce monoclonal antiglobulin serum
Principle of the Antiglobulin Test
- Application of the DAT is for detection of Hemolytic Disease of the newborn (HDN), Hemolytic Transfusion Reaction (HTR), and Autoimmune Hemolytic Anemia and Drug Induced
Evaluation of a Positive DAT
- Interpretation requires knowledge of the patient diagnosis, drug therapy, and recent transfusion history
- To complete investigations of DAT, there should be an answer to "Is there evidence of in vivo hemolysis?"
DAT Investigations
- Has the patient been transfused recently?
- Does the patient's serum contain unexpected antibodies?
- Is the patient receiving any drugs?
- Has the patient received blood products or components containing ABO-incompatible plasma?
- Is the patient receiving anti lymphocyte globulin or antithymocyte globulin?
- Is the patient receiving IVIG or IV RhIG?
IAT- Indirect Antiglobulin Test
- The principle is that it is performed to determine in-vitro sensitization of RBC
- Applications are for compatibility testing, antibody screen, phenotype, and titration
Factors Affecting the Antiglobulin Test
- The ratio of serum to cells should increase serum to cell increase sensitive 40:1
- Albumin "bovin albumin" decreases incubation time and allows antibody-coated cells to come into closer contact
- LISS enhances antibody uptake and allows incubation time to be decreased
- Polyethelene Glycol (PEG) increases antibody uptake, by using with AHG to avoid false positive, and with LISS increase detection of clinically significant antibody and decreases detect of clinically insignificant antibody
Temperatures
- The optimal temperature for IgG is 37°C
Washing of RBCs
- Washing of RBCs is to remove free unbound serum globulins
- Should be in as short time as possible to minimize the elution of low-affinity antibodies and used saline 7.2-7.4 PH should be as fresh as possible
Centrifugation for reading
- Uses between 500-1000 relative centrifugal forces for 15-20 seconds and depends on cell suspension
- The IAT detects in-vitro sensitization of RBCs and can be applied to compatibility testing, antibody screen, antibody identification, RBC phenotyping, and titration studies
- A positive DAT is followed by a DAT panel using monospecific anti-IgG and anti-C3d, and EDTA should be used to collect blood samples for the DAT
Low ionic polybrene technique
- Low ionic solutions are used to rapidly sensitize cells with antibody
- Polybrene is a potent rouleaux-forming reagent to allow sensitized cells to approach each other to permit cross-linking by the attached antibody
- Use high-ionic strength solution to reverse the rouleaux
Enzyme-Linked Antiglobulin Test (ELAT)
- The number of IgG molecules per RBC can also be determined from this procedure
Solid Phase
- Solid phase technology by different techniques either test tubes or microplates and can perform direct and indirect test
The Gel Test
- This is a process to detect RBC antigen-antibody reaction by using a chamber filled with polyacrylamide gel that acts as a trap for agglutinated RBCs
Gel test types
- Neutral gel doesn't contain any specific reagent for Antibody screen by enzyme or Reverse grouping
- Specific gel test uses a specific reagent for Antigen determination
- Low ionic antiglobulin test (GLIAT) is used for IAT and DAT, Compatibility test, and Phenotype
- Detection of antibody by GLIAT provides a safe, reliable, and easy-to-read AHG test.
- Cards should be centrifuged at 910rpm for 10min
- Elution of low-affinity antibody avoided by gel tech becuase "there is no washing steps"
Key points
- A positive DAT is followed by a DAT panel using monospecific anti-IgG and anti-C3d to determine the specific type of protein sensitizing the RBC
- EDTA should be used to collect blood samples for the DAT to avoid in-vitro complement attachment associated with refrigerated clotted specimens
- AHG reagents containing anti-IgG are needed for the detection of IgG antibodies because the IgG monomeric structure is too small to directly agglutinate sensitized RBCs
- Polyspecific AHG sera contain antibodies to human IgG and the C3d component of human complement
- Monospecific AHG sera contain only one antibody specificity: either anti-IgG or antibody to anti-C3b-C3d
- The DAT detects in-vivo sensitization of RBCs with IgG and/or complement components and Clinical conditions that can result in a positive DAT include HDN, HTR, and AIHA
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