DAT & IAT Flashcards
What is the purpose of the Antiglobulin Test?
To detect cells that have become coated with antibodies and/or complement
Often referred to as the Coombs test.
What types of antihuman globulin reagents are available?
- Monospecific AHG
- Polyspecific AHG
What does Polyspecific AHG contain?
- Antibody directed against human IgG antibody
- Human complement – C3d complement component
What does Monospecific AHG contain?
- Anti-IgG only
- Anticomplement (anti-C3b or anti-C3d)
What is another name for Anti-Human Globulin (AHG)?
Coombs serum
What is a critical step in the DAT/IAT testing procedure?
Cell washing is a critical step in the procedure
What can inadequate washing during the DAT procedure result in?
Neutralization of AHG reagent, leading to false negative results
What conditions can cause a positive DAT?
- Hemolytic Disease of the Fetus and Newborn (HDFN)
- Transfusion Reaction
- Autoimmune Hemolytic Anemia (AIHA)
- Viral pneumonia
- Infectious mononucleosis
- Infectious hepatitis
- Myeloblastic Anemia
- Acquired hemolytic anemia
What information must be obtained when a positive DAT occurs?
- Diagnosis
- Medications
- Recent transfusion history
- Other lab values that may indicate RBC destruction
What is the procedure for performing a Direct Antiglobulin Test (DAT)?
Wash patient red cells 3 to 4 times with physiologic saline, add AHG reagent, and interpret agglutination
What is the principle of the Indirect Antiglobulin Test (IAT)?
Patient’s serum is tested for presence of clinically significant antibodies using an indirect antiglobulin method
What are the uses of the antibody screen in IAT?
- Detect unexpected alloantibodies
- Identify auto-antibodies
- Pre-transfusion testing
- Prenatal blood work
What can cause rouleaux formation in a patient’s serum?
Altered albumin to globulin ratio in serum or administration of plasma expanders like dextran
How can you differentiate between true agglutination and rouleaux?
Rouleaux disperses with the addition of saline, while true agglutination does not
What are the limitations of the antibody screen?
- Low frequency antigens may not be detected
- Low antibody titres may not be detected
- Complement dependent antibodies may not be detected
What is the primary difference between DAT and IAT?
DAT detects in-vivo attachment of antibodies to red cells, while IAT detects in-vitro attachment
What is the role of Coombs control cells?
Used in AHG testing to confirm the presence of antibodies
What are IgG-Sensitized Cells used for in AHG testing?
They are required as a control system when AHG results are negative
When added to a negative AHG test, they should cause agglutination.
How are Coombs Control Cells prepared?
By sensitizing group O Positive cells with anti-D antisera
Sufficient anti-D antisera is added to coat the red cells.
What does a negative Coombs control result indicate?
AHG not added, AHG was inactive or neutralized, incorrect procedure followed
This includes issues like incubation time, temperature, washing, and cell suspension concentration.
What can cause false negative results in Coombs testing?
Failure to add AHG, inadequate washing, delay in adding AHG, presence of fibrin clots
Other factors include dirty glassware, over or under centrifugation.
Fill in the blank: Coombs Control Cells are cells coated with _______.
IgG AB
True or False: Inadequate cell washing can lead to unbound antibodies remaining in the RBC suspension.
True
What does a positive Coombs Control result indicate?
AHG was added and is detecting antibody globulin adsorbed to Coombs Control Cells
This verifies that the test is valid.
What is the lifespan of transfused cells?
90 days
RBCs have a lifespan of 120 days, but transfused cells are considered to have a shorter lifespan.
What is the significance of the IS phase in antibody testing?
Cold (IgM) reactions may occur at lower temperatures
IS stands for Immediate Spin.
What type of antibody is most probably present when there is a consistent strength in reactions?
Single alloantibody
What does a reaction in all cells indicate?
Possible autoantibody
What does the rule of three confirm?
Presence of the antibody
A p-value ≤ 0.05 must be observed for confirmation.
What are the requirements to fulfill the rule of three?
- Positive with 3 cells with the antigen
- Negative with 3 cells without the antigen
What is red cell antigen typing?
Patient cells plus reagent antiserum
What technique is used for antigen typing for weak D and other clinically significant antibodies?
IAT technique
What must always be run when performing red cell antigen testing?
Known positive and negative controls
What is the purpose of a positive control in red cell antigen testing?
Verifies antiserum is acting properly
What is elution in the context of antibody testing?
Removal of antibody bound to the red cells
What is an eluate?
Supernatant resulting from an elution test
What is the difference between absorption and adsorption?
- Absorption: Removal of unwanted antibody
- Adsorption: Adding a specific antigen to remove antibody
What is the role of enzymes in antibody identification?
Alter reaction conditions to assist in AB identification
What is the one-stage method of enzyme treatment?
Simultaneous incubation of patient serum, enzyme, and red cells
What is the two-stage method of enzyme treatment?
Panel cells are pre-treated with enzyme before testing
What do sulfhydryl reagents do?
Cleave disulfide bonds of IgM molecules
What is the purpose of neutralization in antibody testing?
Inactivation of an antibody by binding with a soluble antigen
What substances can be used for neutralization?
- Serum/plasma for Anti-Lewis, anti-Chido, Anti-Rodgers
- Hydatid cyst fluid for anti-P1
- Urine for Sd
a - Breast milk for anti-I
What is the expected outcome of a control in the neutralization technique?
Control should remain positive
What indicates a single warm alloantibody in testing results?
Antigen present on both SI and SII
What is the most probable antibody type indicated by a reaction strength of 2+ in AHG?
Warm alloantibodies
What is the implication of performing additional testing to meet the rule of three?
To confirm the presence of multiple antibodies
What is the significance of using selected cells in antibody identification?
To confirm or eliminate the antibody
What must selected cells be negative for?
The known antibody
What is the purpose of neutralization in antibody detection?
To eliminate cold antibodies that may mask the presence of clinically significant antibodies
Neutralization is crucial for accurate antibody identification in blood banking.
What are Lewis substances?
Le a and Le b substances present in saliva, used to neutralize Lewis antibodies in patient specimens
Blood banks often use commercially prepared Lewis substances.
What is the source of Sd a blood group substance?
Present in soluble form in urine
Urine is added to patient serum to neutralize anti-Sd a.
What is the significance of P1 substance in antibody detection?
Neutralizes anti-P1, allowing detection of underlying alloantibodies
P1 substance is found in hydatid cyst fluid and pigeon eggs.
What are the two types of autoantibodies?
Cold and warm reacting autoantibodies
Differentiation is crucial as they can mask significant alloantibodies.
What causes a positive DAT in warm autoantibodies?
IgG antibodies coating the red cells
Most panel or screening cells will be positive in these cases.
What is WAIHA?
Warm autoimmune hemolytic anemia
Caused by warm autoantibodies that react with red blood cells.
How can cold autoantibodies be identified?
When all screening cells, panel cells, and autocontrol are positive at IS and weaken with incubation
Cold autoantibodies are typically IgM and may fix complement.
What is the purpose of prewarmed techniques?
To resolve cold autoantibodies during testing
Red cells, serum, and saline are incubated at 37°C before combining.
What is adsorption in the context of antibody detection?
Removing antibody from serum by adsorption to RBCs carrying corresponding antigens
This aids in identifying underlying antibodies.
What is the main method used for RBC adsorption?
Using autologous red cells or allogenic red cells to remove autoantibody
Autologous cells can only be used if the patient has not been transfused or pregnant in the past 3 months.
What is the goal of cold autoadsorption?
To remove cold reactive autoantibodies for detecting clinically significant alloantibodies
It involves incubating the patient’s RBCs and serum plasma at 0-5 °C.
What indicates the need for autoadsorption methods?
Positive DAT result not caused by a transfusion reaction or HDN
Also used when both DAT and IAT are positive.
What must be done during allogenic adsorption?
Use phenotypically matched or differential adsorptions to remove autoantibodies
This is necessary when autologous cells are insufficient.
What is the ZZAP method of adsorption?
A combination of proteolytic enzyme (papain) and DTT used for autoantibody removal
It destroys Kell, M, N, S, and Duffy antigens.
What does a negative DAT indicate after the adsorption treatment?
No autoantibody remains on the RBCs, allowing for alloantibody testing
If the serum reacts with group O screening cells, an alloantibody is present.
What are high-frequency antigens?
Antigens present in most individuals (>98%)
Examples include U and HTLA antigens.
What is the main challenge in finding compatible blood for patients with high-frequency antibodies?
Difficulty finding compatible blood due to the prevalence of these antigens in the population
Autologous donations are encouraged in these cases.
What are high frequency antigens?
High frequency antigens occur in most individuals (>98%)
Examples include U and HTLA (Ch, Rg).
When should low frequency antigen antibodies be suspected?
When the crossmatch is incompatible and no problems occur with ABO or DAT
Antibodies to low frequency antigens occur in <10% of the population.
What is the purpose of the prewarmed technique?
To eliminate non-significant antibody reactions and identify clinically significant antibodies
IgM antibodies usually have a thermal range below 20°C.
What is the direct antiglobulin test (DAT)?
A test for the in vivo coating of RBCs with antibody or complement
AHG is added to washed patient red cells to determine if IgG or complement are coating the RBCs.
What information can a positive DAT provide?
- Recent transfusion may indicate alloantibody coating transfused cells
- No transfusion may indicate autoantibody coating own cells
- In vitro bound complement may occur with a red top tube
Confirm by using an EDTA specimen.
What is the purpose of elution techniques?
To free antibodies from sensitized red cells for identification
Elution is performed when DAT is positive.
What is an eluate?
The term used for the removed antibodies after elution
It is useful in positive DAT investigations.
List some uses of the eluate.
- Investigate suspected acute or delayed hemolytic transfusion reactions
- Investigate fetal-maternal incompatibility
- Investigation of a positive DAT
- Detect weakly expressed antigens
- Identify multiple antibodies
- Prepare antibody-free intact RBCs for phenotyping or autoabsorption
What are the key objectives of elution techniques?
- Release antibodies
- Concentrate antibodies
- Purify antibodies
What is total elution?
Antibody is released and the RBCs are rendered useless for identification
This is required for antibody identification.
What is partial elution?
The antibody is removed while the RBCs remain intact
This allows for phenotyping or preparation of autoabsorption.
What are some methods of elution?
- Heat at 56°C
- Freeze-thaw
- Cold acid
- Digitonin acid
- Dichloromethane
- Chloroform
- Xylene
- Ether
- Elution using R–E–S
What is the significance of using CDP and glycine acid EDTA reagents in elution?
They can dissociate IgG from the RBC without damaging the antigens
This is useful if the RBC needs to be antigen typed.
