ANTIGLOBULIN TESTING Flashcards
It obtained from immunized nonhuman species bind to human globulins such as IgG or complement either free in serum or attached to antigens on red blood cells (RBC’s).
ANTIHUMAN GLOBULIN TEST
It detect IgG or complement-sensitized RBCs?
ANTIHUMAN GLOBULIN TEST
What is the 2 MAJOR TYPES OF BLOOD GROUP ANTIBODIES
IgM, IgG
Bind to corresponding antigen and directly agglutinate RBCs suspended in saline
IgM
It is a pentamer, and is considered to be the best agglutinin
IgM
“nonagglutinating” or “incomplete antibodies” – because their single monomer structure is too small to directly agglutinate sensitized RBCs
IgG
precipitin so it produces small clumps as compared to agglutination.
IgG
1945, they described the use of the antiglobulin test for the detection of weak and nonagglutinating Rh antibodies in serum.
Coombs, Mourant, and Race
1946 ,Coombs and coworkers described the use of ?
AHG to detect in vivo sensitization of the RBCs of babies suffering from HDFN.
In 1908, Moreschi described the principle of the
Anti-globulin test
In 1908 - Moreschi described the principle of the Anti-globulin test which included what blood group antibodies and associated antigen.
Kell blood group antibodies and associated antigen
Injection of human serum into rabbits to produce antihuman serum
Coombs procedure
In Coombs procedure the one we see in this procedure is?
Hemagglutination
Result of RBCs has been sensitized by an antibody
Hemagglutination
In 1947 - Coombs and Mourant demonstrated that the antibody activity that detected
Rh
Coombs and Mourant demonstrated that the antibody activity that detected Rh
o antibodies were associated with the ?
anti-gamma globulin fraction
In 1951 (?) Observed that different reaction patterns were obtained when dilutions of AHG were used to test cells sensitized with warm as compared to cold antibodies.
Dacie
Warm reacting antibodies
IgG
Cold reacting antibodies
IgM
The Antiglobulin test can be used to detect RBCs sensitized with?
IgG alloantibodies, IgG autoantibodies, and complement components.
Sensitization can occur either in?
Vivo and vitro
Ag-Ab binding happened inside the body? In vivo or in vitro?
vivo
outside the body? In vivo or in vitro?
vitro
is detected by a one stage procedure because their already sensitized RBC in body.
vivo sensitization
vivo sensitization is detected by?
Direct antiglobulin test (DAT)
The use of AHG to detect in (?) sensitization of RBCs is a two-stage technique.
vitro sensitization
vitro sensitization is detected by
Indirect antiglobulin test (IAT)
What is the 2 procedures in vitro sensitization?
The 2 procedures:
- We need to incubate them to 37 degrees Celsius because they are IgG.
- Add you anti-human globulin reagent to demonstrated if they can agglutinate.
Defined by FDA and CBER (Center for Biologics Evaluation and Research)
ANTIHUMAN GLOBULIN REAGENTS
FDA and CBER transcribe?
Food and Drug Administration, Center for Biologics Evaluation and Research
ANTIHUMAN GLOBULIN REAGENTS 2 types?
POLYSPECIFIC, MONOSPECIFIC
is the one we check because it presents in all pathway.
- C3
Antibody to human IgG and to the C3d component of the human complement, contains more than 1 type of antibody.
POLYSPECIFIC
POLYSPECIFIC commonly used?
Anti-C3b, anti- C4b and anti-C4d may also be present
- contain only one antibody specificity: either anti IgG or antibody to specific complement components (C3b or C3d)
MONOSPECIFIC
Monospecific commonly used?
: anti-IgG and anti-C3b, anti-C3d
o No anticomplement activity
o Contain antibodies specific for the Fc fragment of the gamma heavy chain of the IgG molecule.
o IgG will bind to Fc portion
Anti-IgG
Anticomplement reagents?
Anti-C3b and anti-C3d reagents
Often a blend of monoclonal anti-C3b and monoclonal C3d
- Anticomplement
Complement is the start of the reaction that is common in 3 pathways
classical, alternative, mannose)
foreign antigen to the rabbits
- Human globulin
- Many Ag to bind
POLYCLONAL ANTIBODIES
- Mixture of antibodies from different plasma cell clones.
- Directed against multiple types of epitopes.
POLYCLONAL ANTIBODIES
determinant site that is found in the antigen to be able to recognize by the antibody
o Epitopes
- 1 Ag can only be bind.
- Derived from one clone of plasma cells and recognize a single epitope.
MONOCLONAL ANTIBODIES
can be used to produce monoclonal antiglobulin serum
- Hybridoma technology
POLYCLONAL AHG PRODUCTION usually prepared using?
rabbits
IN POLYCLONAL AHG PRODUCTION, Large volumes of antibody are required (?) and (?) may be used
Sheep or Goats
- If the rabbit is injected with a complement, it will produce
Monospecific Polyclonal Anti-C3b, Anti-C3d.
- If the rabbit is injected with an IgG, it will produce
Monospecific Polyclonal Anti-IgG.
When we mixed both Monospecific Polyclonal Anti-C3b, Anti-C3d and Monospecific Polyclonal Anti-IgG together, it can produce
Antihuman Globulin Polyspecific Polyclonal Blend
means there is only 1 Ab but can detect many epitopes (reacts)
Monospecific Polyclonal
- Recognize multiple epitopes on any one antigen.
POLYCLONAL ANTIBODIES
contains a heterogenous complex mixture of antibodies of different affinity.
- Serum obtained
made up mainly of IgG subclass. (4 subclasses of IgG)
- Polyclonals
- often used to generate polyclonal antibodies that target unique epitopes, especially for protein families of high homology
- Peptide immunogens
MONOCLONAL AHG PRODUCTION also called?
hybridoma technology
MONOCLONAL AHG PRODUCTION was discovered by?
Kohler and Milstein
in monoclona AHG production, Immunization of laboratory animals usually use is (?) with purified human globulin.
mice
For HYBRIDOMA to form we need to incubate it to
HAT medium (hypoxanthine–aminopterin–thymidine)
IN PREPARING ANTI COMPLEMENT in MONOCLONAL AHG PRODUCTION we will have?
Monoclonal anti-C3b, d, or g (1 only Ab target)
IN PREPARING ANTI-IGG in MONOCLONAL AHG PRODUCTION we will have?
Monoclonal ant-IgG (1 only Ab target)
If we mix the anti-C3b,d,g and anti-IgG, we will have an
anti-human globulin (AHG) polyspecific monoclonal blend
- Contains only one antibody specificity
MONOSPECIFIC AHG
is produced as a monoclonal, polyclonal or blended formula.
- Monospecific anti-IgG
- Anti-IgG: Majority are mixture of (?) and (?) subclasses
IgG1 and IgG3
- High specificity
- Detects only one epitope on the antigen.
- Consist of only one antibody subtype (e.g. IgG1, IgG2, IgG3, iGG4)
MONOCLONAL ANTIBODIES
- Antibody molecules and complement components are
globulins
reacts with human globulin molecules, either bound to RBCs or free in serum. Washed RBCs coated with human globulin are agglutinated.
AHG
Detects in vivo sensitization of RBCs with IgG or complement components. Clinical conditions that can result in in vivo coating of RBCs with antibody or complement
- DAT
Clinical conditions that can result in in vivo coating of RBCs with antibody or complement are:
o HDN
o HTR
o Autoimmune hemolytic anemia and drug-induced hemolytic anemia
RBC have metabolites of the drugs attached which usually cause by ?
ibuprofen
o One drop of 3% to 5% suspension of washed RBCs with polyspecific (anti-IgG, anti-C3d) reagents.
- DAT Panel
Anti-IgG (+) and Anti-C3d (+)
67% of Warm Autoimmune Hemolytic Anemia
Anti-IgG (+) and Anti-C3d (-)
20% Warm Autoimmune Hemolytic Anemia cases
Anti-IgG (-) and Anti-C3d (+)
(13%) of Cold Hemagglutinin disease (CHD), Paroxysmal Cold Hemoglobinuria (PCH), Warm Autoimmune Hemolytic Anemia (WAIHA)
In vivo sensitization: Maternal Ab coting fetal RBC
HDN
In vivo sensitization: Recipient Ab coating donor RBC
HTR
In vivo sensitization: Autoantibody coting individual RBC
AIHA
In Hemolytic Disease of the Newborn (HDN), what we check for DAT will be
Fetal RBC
In Hemolytic Transfusion Reaction (HTR) we will be checking the
Recipient blood
- Own antibodies are attacking its own cell
Autoantibody
- performed to determine in vitro sensitization of RBCs and is used in the following situations
INDIRECT ANTIGLOBULIN TEST (IAT)
o Detection of incomplete (non-agglutinating) antibodies to potential donor RBCs (compatibility testing) or to screening cells (antibody screen) in serum
INDIRECT ANTIGLOBULIN TEST (IAT)
Ab detection
copatibility testing, Ab screening
Ab identification
Ab panel
Ab titrataion
Rh Ab titer
RBC phenotype
RBC Ag detection
- Cells suspended in saline:
INCUBATION TIME
incubation times vary between (?) if there is no reaction medium
30-120 minutes
shortened the Incubation time to 10 to 15 minutes
Low-Ionic-Strength Solution, Polyethylene Glycol