(M) Antihuman Globulin Testing Flashcards
- aka immuno globulins
- complex protein made by plasma cells
- binds to foreign molecules such as an antigen
antibody
what complement pathway does the antigen-antibody binding activate?
classical pathway
When an antibody is able to agglutinate an antigen, it is
called ____________
agglutinin
Identify the immunoglobulin class
● 80% in the total circulation
● Clinically significant as they react at body
temperature
● Crosses the placenta due to its small size
● MW: 150 kd
IgG
Identify the immunoglobulin class
● 6% in the total circulation
● Naturally occurring
● Biggest immunoglobulin (pentameric)
● MW: 900 kd
IgM
Identify the immunoglobulin class
● 13% in the total circulation
● Major immunoglobulin found in body
secretions
● MW: 160 - 500 kd
IgA
Identify the immunoglobulin class
● 1% in the total circulation
● Found in the cell membrane Ig particularly in B
cells
● MW: 180kd
IgD
Identify the immunoglobulin class
● Less than 1% in the total circulation
● Mediates allergic, hypersensitivity reaction
● MW: 196 kd
IgE
What is the principle of antiglobulin test / Coomb’s Test
Detect cells that have become coated with antibodies or complement
- What is the color of Coomb’s serum
- color of monospecific anti-complement
- green
- colorless
The IgG antibody directed against human immunoglobulin or antibodies, and/ or complement
- produced by injecting a rabbit with human antibodies
AHG
Type of AHG
contains only one antibody specificity either anti-IgG or anti-complement
monospecific
Types of AHG
contains both anti-IgG and anti-complement
polyspecific
Complete the statements
- An __________ is a blend of monoclonal anti-C3D.
- An ________ contains antibodies to the Fc fragment (gamma HC) of the IgG molecules
- anti-complement
- anti-IgG
Identify the following:
- antibodies derived from one clone of plasma cells and recognize a single epitope
- mixture of antibodies from different plasma cell clones
(monoclonal)
polyclonal antibodies
What are the globulins that AHG may be directed against?
○ IgG
○ IgM
○ IgA
○ C3
reminder: GMA is channel 3
animal is injected with either human antigen or IgGs
a. conventional method
b. hybridoma
c. both
d. neither
both
what do the rabbits produce after being injected with either human complement or IgG?
either:
* monospecific polyclonal anti-C3b
* monospecific polyclonal IgG
what is produced after the combination of monospecific polyclonal anti-C3b and monospecific polyclonal OgG
AHG polyspecific polyclonal blend
once the antibodies created using the conventional method are collected, it will be purified and adsorbed with O, A, and B red cells to remove what?
heterospecific antibodies that may cause positive results in testing
What is the disadvantage of the conventional method ?
dependent on the lifespan of the inoculated animals
In hybridoma technology, after the antibody producing lymphocytes are collected from the spleen of the inoculated mouse, it is fused with what cells to form hybridoma?
cancer / myeloma cells
T or F
when hybridoma are already present, they can now be cultured for the clones to grow in tissue cultures to yield a pure monoclonal band of the antibodies, resulting to a finite supply of the antibodies
f (resulting to an indefinite supply of the antibodies)
What is the disadvantage of the hybridoma technology?
only recognizes one single epitope
Hybridoma technology
what happens when you combine the anti-complement and the anti IgG ?
polyspecific monoclonal blend
FDA licensed antihuman globulin reagents
POLYSPECIFIC
a. rabbit polyclonal
b. rabbit / immune monoclonal blend
- Contains anti-IgG and anti-C3d (may contain other anti-complement and other anti- immunoglobulin antibodies)
- Contains a blend of rabbit polyclonal anti- human IgG and Anti-C3d is a murine monoclonal IgM antibody
- a
- b
FDA licensed antihuman globulin reagents
MONOSPECIFIC
a. Anti-IgG (Rabbit polyclonal)
b. Anti (Gamma-done AHG)
c. Anti-C3d
- Contains anti-IgG with no anti-complement activity
- Murine monoclonal IgM antibody secreted by a hybridoma cell line
- Causes the agglutination of RBCs coated with human C3d and/or C3b complement components
- A
- B
- C
What does polyscpecific AHG reagent contain?
human IgG and C3d
a. monospecific AHG rgt
b. polyspecific AHG rgt
- routinely used in blood bank
- causes false negative with anti-IgG
- causes false positive
- No interference from clinically insignificant IgM-complement fixing antibodies
- A
- A
- B
- A
a. monospecific AHG rgt
b. polyspecific AHG rgt
- uses the pre-warm technique when a positive result is yielded
- has high cost with high repeats
- b
- b
Action of AHG except:
a. binds with human globulin
b. acts as a bridge, combining the Fab region of a sensitizing antibody
c. forms a lattice once attached to IgG, creating agglutination
d. Must contain anti-IgG
b (combining the Fc region of a sensitizing antibody)
Coombs control cells or check cells
- checks the validity of the result when AHG reagent is used
- contains RBCs coated with IgMs
a. only statement 1 is true
b. only statement 2 is true
c. both statements are true
d. neither of the statements are true
a
Coombs control cells or check cells
- It is composed of Rh positive cells coated with anti-d antibodies
- It is coated with the C3 portion of the complement
a. only statement 1 is true
b. only statement 2 is true
c. both statements are true
d. neither of the statements are true
c
Coombs control cells or check cells
- Coombs control cells will react with the antigen in the AHG reagent
- In preparing check cells, O negative red cells are used with the addition of anti-D reagent.
a. only statement 1 is true
b. only statement 2 is true
c. both statements are true
d. neither of the statements are true
d.
- check cells will react with the ANTIBODY of the reagent
- In preparing check cells, O POSITIVE red cells are used
What should be the result of the binding of IgG coated RBCs + AHG and check cells
positive
The following are true of what Coombs control cells prove except
a. Coomb’s rgt was added
b. Coomb’s rgt was active, potent, not expired and is working properly
c. the wash step was adequate to remove any unbound antibodies
d. If agglutination was not present, this indicates a true negative result.
d. if agglutination is present, this indicates a true negative result
What are the two methods of anti globulin testing
- direct antiglobulin test
- indirect antiglobulin test
DAT or IAT?
- detects antibody sensitization IN VITRO
- detects antibody sensitization IN VIVO
- sample used is serum or plasma
- sample used is the patient’s red cells
- A one-step procedure
- IAT
- DAT
- IAT
- DAT
- DAT
READ
DAT TUBE METHOD PROCEDURE
- Get an EDTA sample from the patient.
- Prepare 3-5% patient red cell suspension.
○ Patient’s red cells are washed with 0.9% NaCI a minimum of 3 times to remove plasma that may contain unbound antibodies.
○ Wash it, then obtain the required volume. You can prepare 1ml (one drop of washed patient’s red cell) with 9 drops of your NSS. - Place 1 drop of 2-5% patient washed RBC.
- Add 2 drops of AHG reagent.
- Tube is centrifuged.
- If IgG or C3 is coating the cells, agglutination will occur
(POSITIVE TEST).
○ ThiswilldependonthetypeofAHGreagentusedi.e., if C3 is coating the cell, and monospecific anti-IgG AHG reagent is used, there will be no agglutination.
○ If neither is present, there will be no agglutination (NEGATIVE TEST). - Each negative test is validated (controlled) through the addition of Coombs Control Cells or check cells
○ Coombs Control Cells are only added for all negative results. It is not necessary for positive results because, if it is positive, it means there is already the presence of antibodies attached to the patient’s red cells.
○ After the addition of check cells in negative results, you should obtain a positive result. (this validates the negative result obtained prior to adding of check cells)
DAT (TUBE METHOD) PROCEDURE IDENTIFICATION
- What anticoagulant is used?
- RBC suspension percentage
- What is reagent is used to wash the red cells?
- how many drops of washed RBC is used?
- how many drops of AHG reagent is used?
- EDTA
- 3-5% red cell suspension
- 0.9 NaCl
- 1 drop
- 2 drops
If IgG or C3 is coating the cells agglutination (will/will not) occur
will occur
agglutination = positive test
T or F
Agglutination will depend on the type of AHG reagent used i.e., if C3 is coating the cell, and monospecific anti-IgG AHG reagent is used, there will be no agglutination.
T
T or F
If either IgG or C3 is present, there will be agglutination
T
T or F
Coombs Control cells are added for both negative and positive results
F (not necessary for positive results because this indicates the presence of antibodies attached to the patient’s red cells)
what are the three conditions that result to a positive DAT?
- HDFN (hemolytic disease of the fetus and newborn)
- HTR (hemolytic transfusion reaction)
- AIHA (autoimmune and drug induced hemolytic anemia)
What causes a positive DAT reaction in HDFN?
infant’s cells are coated with maternal IgG
What causes a positive DAT reaction in HTR?
patient’s antibody coats the donor antigen cells
a. acute hemolytic transfusion reaction
b. delayed hemolytic transfusion reaction
c. both
d. neither
- complement is activated -> donor red cells are lysed
- DAT is positive due to IgG
- gradual drop in hemoglobin in 2-10 days
- TRALI
- a
- c (acute hemolytic transfusion may be positive due to IgG or complement, delayed = only IgG)
- b
- a
familiarize the mechanisms of AIHA
- Drug adsorption
- immune complex
- induction of autoimmunity
- membrane modification
familiarize the diseases caused by AIHA that may cause autoantibody to coat the patient’s red cells
- cold hemagglutinin disease
- warm autoimmune hemolytic anemia
- paroxysmal cold hemoglobinuria
mechanism of AIHA
o Drug attaches to RBC directly
o Antibody directed at drug only
o DAT positive due to IgG produced by patient
because they recognized the drugs as foreign
creating antibody against it
Drug adsorption
mechanism of AIHA
o Antibody is directed at a “neoantigen” having determinants on both the drug and the red cell membrane
o Antibodyactivatescomplement
o DAT positive due to complement attached to the
red cell
Immune complex
mechanism of AIHA
o Drug modifies red cell membrane resulting in production of autoantibody (antibodies against your own red cells)
o DAT positive due to IgG and occasionally complement
Induction of autoimmunity
mechanism of AIHA
o Nonspecific adsorption of plasma proteins (non- immunologic)
o DAT will be positive due to whatever has “stuck” to the membrane – IgG, complement, IgM, IgA
Membrane modification
READ!
IAT (TUBE METHOD) PROCEDURE
- Add one or two drops of plasma or anti-serum containing antibody to a test tube
- Add one drop of red cells (antigen source) to the tube.
o Optimum plasma to red cell ratio → 2:1 - The tube is incubated at 37C. The length of incubation is dependent on the medium.
o Enhancement reagents may be added before or during
incubation to increase sensitization and agglutination,
thereby enhancing the reaction.
o Incubation is done to enhance reaction as most IgGs
have their optimum temperature of reactivity at body
temperature - After incubation, centrifuge and read then the cells are washed with saline a minimum of 3 times, to remove any unbound antibodies.
o Unbound antibodies, if not removed, will neutralize the
AHG reagent, causing a false negative result - Following the final wash, two drops of AHG reagent are added to the dry cell button. The tube may be read microscopically, depending on the test medium.
- Coombs control cells are added to each negative test. The tubes are centrifuged and results are read.
IAT (TUBE METHOD)
- how many drops of plasma or anti-serum containing antibody to a test tube?
- how many drops of red cells needed in the tube?
- what is the optimum plasma to red cell ratio?
- at what temp is the tube incubated at?
- 2
- 1
- 2:1
- 37C
IAT (TUBE METHOD) PROCEDURE
- the cells are washed with what reagent?
- how many times are the cells washed?
- how many drops of AHG reagent is needed?
- excess washing reagent would dilute the AHG reagent which leads to what type of result?
- Saline
- 3 times
- 2 drops
- False negative
What is the purpose of centrifugation and incubation in the IAT procedure?
enhance reaction of the IgG with the antigens present in the red cell
provide the sample used in the following tests:
- IAT gel method
- IAT tube method
- DAT
- plasma
- serum
- red cells
Provide the sources of antigen for the following tests:
1. crossmatching
2. antibody screening
3. antibody detection
- donor’s red cells
- and 3. commercially prepared reagent cells
APPLICATIONS OF IAT
* Detects antibodies in the patient’s serum.
* Uses reagent red cells as a source of known antigen.
* The reagent source is commercially prepared reagent
cells and it comes with a panel sheet which has the antigrams. You’ll see there what antigens are present on the red cells.
o Ifyouknowhowtodocrossmatch,thenthat’sthesame
principle as antibody screening and antibody identification or panel testing.
ANTIBODY SCREEN
APPLICATION OF IAT
- Identifies antibodies
- Uses reagent red cells as a source of known antigen
- Once antibody screening is positive, proceed with
identification to identify the particular antibody is present. - The antigen source is also commercially prepared reagent
cells. - It comes with 10-11 panels / vials of red cells with known
antigens.
Antibody identification or antibody panel
APPLICATIONS OF IAT
- Determines patient’s compatibility with donor
- Uses donor red cells (antigen source) and patient’s
serum (antibodies) - Usually performed only when a patient has an antibody or a history of exposure to antigens, sensitizing the patients to produce antibodies that may be because of blood transfusion or pregnancy.
Antiglobulin crossmatch
study the comparison of AHG methodologies
thanks
enhancement reagents are reagents that adjust the test environment thereby enhancing the attachment of antibodies if they are present.
potentiators
the only saline agglutinating antibodies
IgM
What are the three actions of potentiators
- reduce zeta potential
- promote agglutination
- enhance antibody uptake
- This refers to the negative surface charge surrounding the RBCS which attract cations such as Na+.
- It is the electrical potential between the red cell surface and the outer ionic cloud.
- This promotes the formation of antibody bridges between cells which we call agglutination.
zeta potential
DIFFERENT ENHANCEMENT REAGENTS
- High molecular weight protein
- Reduces the zeta potential by dispersing some of the
cations surrounding each negatively charged red cell. - Increases the dielectric constant, defined as a measure of the ability to dissipate a charge.
- Disadvantage: 30 minutes incubation time
- Optimum ratio: 2 drops of serum, 2 drops of 22% (w/v) ___________, and 1 drop of 3% to 5% (v/v) red cells
22% Albumin
DIFFERENT ENHANCEMENT REAGENTS
- Made of NaCl, glycine, and albumin
- MOST COMMON potentiator used in blood banks.
- Creates a low ionic environment
- Lowers the zeta potential
- Promotes antibody uptake by the red cells
- Advantage: Shortens incubation time (10-15 minutes)
- Usual ratio: 2 drops of serum and 2 drops of a 3% (v/v)
suspension of cells in _____. - Can be used as a medium to suspend red cells or can be
used as an enhancement reagent so red cells may be suspended in NSS just add 2 drops of ______ on the test system before incubation
Low ionic strength solution (LISS)
DIFFERENT ENHANCEMENT REAGENTS
- _____________l is suspended in a low ionic strength
medium. (water-soluble polymer) - Action: Removes water from the test system, thereby
concentrating any antibody present. - Antibody uptake is increased.
- Increase detection of clinically significant antibodies, and
decrease detection of clinically insignificant Abs (mostly IgM) - Enhances the detection of IgG
- Disadvantage: ___ may cause false positive results as it
causes cellular aggregation
○ Do not centrifuge after a 37°C incubation. Proceed
immediately to the wash phase, with a minimum of 4
washes performed before adding the AHG reagent - ______ is not the potentiator of choice when the patient has elevated proteins, such as in multiple myeloma. In these cases, LISS is the preferred enhancement.
Polyethylene glycol
DIFFERENT ENHANCEMENT REAGENTS
- Ficin, papain, bromelin, and trypsin are the enzymes
commonly used in blood banking - Proteolytic substances
- Modifies the red cell membrane by removing sialic acid
residues, thereby reducing surface charge. - Decreases zeta potential
- Splits polypeptide chains, which further exposes some
antigens so the antibodies can readily attach to the antigen - May enhance the hemolytic activity of some
complement-dependent antibodies.
enzymes
What are other blood groups destroyed by enzymes?
M,N,S Fy, Xga
What are the other blood groups enhanced by enzymes
Rh, P, Le, Jk, and I
What are the four sources of error in antiglobulin testing
- adequate wash
- centrifugation
- problems with reagents or saline
- problems reading reactions
pls read the causes of false positive and false negative results
gaur
