Antiglobulin Test Flashcards
What is AHG?
-Antihuman globulins bind to human globulins
-uses a xenoantibody (non-self)
-normally immunological response is an antigen-antibody component
-in this case, it is actually antibody-antibody binding or complement antibody binding
-the human antibody is the antigen
What is a globulin?
-a protein of some sort, in this case, either IgG immunoglobulin (antibody) or a specific complement component
How is AHG made: Conventional method
-injecting human serum or globulin into animals such as rabbits
-the rabbit’s immune response produces an antibody to the human globulin
-this produces polyclonal AHG
-pooled donor antigen is injected
-after harvesting the serum of many rabbits is also pooled
-broad reactivity including IgG variants
-limited source amount lifetime of the rabbit
How is AHG made: Hybridoma method
-purified human globulin injected into mouse
-this produces monoclonal AHG
-after immune response, antibody-secreting lymphocytes from the spleen are collected
-the lymphocytes are fused with myeloma cells
-hybridoma clones are grown in tissue culture
-infinite source, high titer, well-defined specificities, controlled potency
-single epitope is not as effective as binding always
How to make polyspecific AHG reagents
-This AHG would have the ability to react with both human antibody and human complement
-it is possible to add polyclonal anti-IgG from a group of rabbits and add it to the polyclonal complement from a different group of rabbits to create a polyspecific polyclonal blend
The immucor polyspecific
-used at SMH
-has three parts (Anti-IgG, Anti-C3d, Anti-C3b)
-each part is monoclonal
-together all three parts make a polyspecific monoclonal blend
polyclonal
antibodies derived from more than one parent cell or group of cells
Monoclonal
-antibody derived from a single ancestral antibody-producing parent cell
polyspecific
reagent that contains more than one AHG, usually IgG and C3d
Monospecific
-reagent specific to one type of globulin (ex: Anti-IgG only)
Why do we need AHG?
-AHG bridges the gap between sensitized cells resulting in visible agglutination
-prior to AHG test, only IgM antibodies could be detected
-IgM is a pentamer, because of its large structure they have more antigen binding sites and can directly agglutinate RBCs suspended in saline
-IgG antibodies are small with fewer binding sites that cannot reach each other to agglutinate, they need help
Affinity
strength of interactions between one epitope and an antibody
Avidity
measure of the overall strength of the antigen-antibody complex, all of the binding available creating good agglutination
*polyclonal reagents have better avidity and can create better agglutination
Sensitization
-A condition of being made sensitive to a substance (antigen) after initial exposure. Results in an immunological response and memory
In vitro
-outside of the living body, as in a lab setting
In vivo
-inside of the living body
What are the two major types of antihuman globulin tests that use AHG reagents?
-IAT
-DAT
Indirect Antiglobulin Test
-test is performed to demonstrate in vitro sensitization
-used to detect antigen-antibody reactions that occur in a tube
-a two-step process that requires a long incubation
Direct antiglobulin test
-testing is performed to demonstrate in vivo sensitization
-used to detect cell sensitization that occurred in the body
-cells are already sensitized, think of it as the antibody is already directly on the cells no incubation is needed
-old school name was the Direct Coombs test
sensitization
-is the coating of red blood cells from the interaction between antibodies and antigens
Agglutination
-is the visible lattice that occurs when many sensitized cells are clumping together
What are some clinical conditions that can result in the in vivo coating of red blood cells? (A positive DAT)
- Hemolytic disease of the newborn: maternal antibody coating fetal RBCs
- Hemolytic transfusion reaction: Recipient antibody coating transfused donor RBC
- Autoimmune and drug-induced hemolytic anemia: Autoantibody coating self RBCs
Steps in DAT
- cells coated in vivo
- washed to remove unbound globulin
- The addition of AHG promotes agglutination after centrifugation
Why should DAT be run in EDTA lavender top specimen?
-EDTA is an anticoagulant that chelates calcium which is needed for C1 activation
-this prevents the in vitro fixation of complement associated with clotted specimens (red top tubes)
-this would be seen as a false positive
*The manufacturer recommends a 5-minute delay before reading the complement portion of the DAT to enhance reactivity
chelates
-to bond with (EDTA bonds with Ca+)
DAT panel
First: wash RBCs with one polyspecific reagent
* If negative you are done
* if positive do panel
Second: washed RBCs with two separate reagents
* monospecific anti-IgG
* monospecific anti-C3d
-the monospecific test tells us what type of protein is coating the RBCs
Saline control
-is performed when the polyspecific antiglobulin serum, Anti-IgG, and Anti-C3d testing are all positive
-this control serves to detect spontaneous agglutination of cells (ex: recognition of aggregates caused by Wharton’s jelly or if agglutinates are the result of “complete” (IgM) auto-antibodies not dissociated during the washing process)
Steps of the saline control
- add 1 drop of patient 3-5% cell suspension
- Wash
- Add 2 drops of saline
- centrifuge and read
What does a positive saline control mean?
-the tests for IgG and C3 components coating the patient cells are not valid, additional investigation must be done
What does a negative saline control mean?
the patient’s red blood cells are not spontaneously agglutination therefore, the reaction being seen in the polyspecific and monospecific IgG and C3 are valid
What is a warm Autoimmune hemolytic anemia associated with?
with an IgG-only positive DAT
What is a cold Autoimmune hemolytic anemia/ cold agglutinin syndrome associated with?
C3d only positive DAT
What happens after a positive DAT test
-some institutions do not perform eluates if the complement is the only positive result in the panel
-at SMH we will always do an eluate when any positive DAT test result is found and there is a history of recent transfusion or no history at all
Eluate: removal of antibody coating the surface of a patient’s red blood cells through physical or chemical means
Steps in IAT
- Incubate RBCs with antisera: allows time for antibody molecule attachment to RBC antigen
- Perform a minimum of 3 saline washes: remove free globulin molecules
- Add antiglobulin reagent: forms RBC agglutinates (RBC Ag + Ab + anti-IgG)
- Centrifuge: accelerates agglutination by bringing cells close together
- Examine for agglutination: interprets test as positive or negative
- Grade agglutination reactions: determine the strength of the reaction
- Add antibody-coated RBCs to negative reactions: check for neutralization of anti-sera by free globulin molecules
* coombs control cells are D-positive RBCs coated with anti-D
Why do we add AHG immediately?
cell-bound IgG may detach from red cells and reduce reaction strength or cause a false negative
The IAT has many applications what are they?
- Antibody screen
- Antibody identification
- Compatibility testing
- Titration
- Phenotyping
- Weak D
Antibody screen
-detects antibodies to RBC antigens in patients serum/plasma
Antibody identification
-additional testing to find the antibodies’ specificity
Compatibility testing
-determines if the Donor RBC unit has antigens that agglutinate in patients’ serum/plasma
Titration
-measuring the strength of the antigen-antibody reaction
Phenotyping
-determines the presence or absence of specific antigens on RBCs using known anti-sera
Weak D test
-specialized phenotyping for D antigen
* weak D and control testing will be falsely positive if the patient has a positive DAT
How can a heavy suspension create a false negative?
-due to post-zone activity resulting in too few antibodies to bind the cells together
IAT- factors that affect testing
- Serum-to-cell ratio
- reaction medium
- temperature
- time of incubation
- washing of RBCs
- Saline for washing
- Addition of AHG
- Centrifugation
IAT- factors that affect testing: reaction medium
albumin: macromolecules allow coated RBCs to get closer
LISS: reduces zeta potential also allowing RBCs to get closer (adding serum can reduce sensitivity counteracting the LISS)
PEG: water-soluble linear polymer, removes water molecules, concentrating antibody, only read at IAT phase not 37
IAT- factors that affect testing: washing of RBCs
*one of the most important steps
-should be done immediately after the incubation phase/reading
-should be done in as short a time as possible
-the cell button must be fully resuspended in the residual saline after decanting before the next wash cycle
IAT- factors that affect testing: saline for washing
-older storage containers can decrease pH
-more acidic saline can elute antibodies from red cells (cause false negative)
IAT- factors that affect testing: addition of AHG
-should be done immediately after washing
-cell-bound IgG may detach from the RBCs if there is a delay in adding AHG
* This is why we use check cells
control cells
-they verify negative results when using AHG in both DAT and IAT testing
-referred to as check cells, coombs control cells, or IgG-sensitized cells
IgG-coated check cells
Rh-positive cells that have been coated with anti-D cells will react with the IgG antibody in the polyspecific AHG reagent and the monospecific IgG reagent
complement coated check cells
- cells will react with the monospecific complement region
- complement control cells are typically only used as control cells for the monospecific complement reagent
What are the most common false negatives?
- inadequate washing
- not adding AHG reagent
What does inadequate washing result in?
-unbound globulin binding to the AHG reagent and then it doesn’t create the bridge we need for agglutination
* to counter that use a cell washer or check cells
What does negative check cells mean
-the test is invalid and must be repeated
What is the most common cause for a false positive?
-contamination of tubes or using improper specimen
List of false positive results
- Over reading
- dirty glassware
- The presence of fibrin in the tube
- cells that have a positive DAT will also yield a positive IAT
- improper specimen
- bacterial contamination of saline used in washing
- polyagglutinable cells (these cells always agglutinate)
List of false negative results
- inadequate or improper washing
- improper testing temperature
- improper centrifuge time or RPM
- not adding the proper reagents at the proper time
- incubation time not long enough
- wrong serum-to-cell ratio or cell suspension percentage
- low pH of saline used for washing
Solid phase technology
-microplate semi-automation
-read reverse from tubes, the solid pellet is negative
Gel test technology
-does not require washing, most common automation
-The solid pellet at the bottom of the column is a negative
-RBCs at the top of the column is a positive
-RBCs spread out throughout the column are positive and are graded weak pos to 4+
Low ionic polybrene technique
-low ionic conditions rapidly sensitize cells through proximity
Enzyme-linked antiglobulin test
-color measured spectrophotometrically
