[11] CHAPTER V LESSON 1 Flashcards
AHG’s obtained from immunized nonhuman species bind to human globulins such as (?] either free in serum or attached to antigens on red blood cells (RBC’s).
IgG or complement
2 major types of blood group antibodies:
IgM
IgG
- bind to corresponding antigen
IgM
directly agglutinate RBCs suspended in saline
IgM
- “nonagglutinating” or “incomplete antibodies”
IgG
because their single monomer structure is too small to directly agglutinate sensitized RBCs
IgG
described the use of the antiglobulin test for the detection of weak and nonagglutinating Rh antibodies in serum.
Coombs, Mourant, and Race
described the use of AHG to detect in vivo sensitization of the RBCs of babies suffering from HDFN.
Coombs and coworkers
described the principle of the test
Moreschi
Kell blood group antibodies and assoc. antigen
Moreschi
Reported weeks after the Coombs had described the test
Moreschi
- RBCs has been sensitized by an antibody
Hemagglutination
Early AHG reagents were prepared using a (?) as the immunogen.
crude globulin fraction
demonstrated that the antibody activity that detected Rh o antibodies were associated with the anti-gamma globulin fraction in the reagent.
Coombs and Mourant
: 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
published data showing that the reactivity of AHG to cells sensitized with warm antibodies resulted from anti-gamma globulin activity, whereas antinongamma globulin activity was responsible for the activity of cells sensitized by cold antibodies.
Dacie and coworkers
was shown to be beta globulin and had specificity for complement
nongamma globulin component
Later studies revealed that the complement activity was a result of
C3 and C4
can be used to detect RBCs sensitized with IgG alloantibodies, IgG autoantibodies, and complement components.
antiglobulin test
can occur either in vivo or in vitro:
Sensitization
is detected by a one stage procedure
In vivo sensitization
In vivo sensitization ex.
Direct antiglobulin test (DAT)
The use of AHG to detect [?] of RBCs is a two-stage technique
in vitro sensitization
In vitro sensitization ex.
Indirect antiglobulin test (IAT)
ANTIHUMAN GLOBULIN REAGENTS
Defined by
Food and Drug Administration (FDA) and Center for Biologics Evaluation and Research (CBER)
- contain antibody to human IgG and to the C3d component of the human complement.
- Polyspecific AHG
Other anticomplement antibodies, such as anti-C3b, may also be present.
- Polyspecific AHG
- contain only one antibody specificity: either anti-IgG or antibody to specific complement components (C3b or C3d).
- Monospecific AHG
Licensed monospecific AHG reagents in common use are
- Monospecific AHG
antiIgG and anti-C3b,-C3d.
Contain no anticomplement activity
a. Anti-IgG
Contain antibodies specific for the Fc fragment of the gamma heavy chain of the IgG molecule
a. Anti-IgG
- reactive against only the designated complement components and contain no activity against human immunoglobulins
b. Anticomplement
Anti-C3b and anti-C3d reagents
Monospecific anticomplement reagents: Often a blend of
b. Anticomplement
monoclonal anti-C3b and monoclonal C3d.
FDA Licensed Antihuman Globulin Reagents
Polyspecific
Monospecific
Anticomplement
Polyspecific
1. Rabbit Polyclonal
2. Rabbit/murine monoclonal blend
Monospecific
1. Anti-IgG (Rabbit polyclonal)
2. Anti-IgG (Gamma-done AHG)
Anticomplement
Anti-C3d
Contains anti-lgG and anti-C3d (may contain other anticomplement and other antiimmunoglobulin antibodies)
Rabbit Polyclonal
Rabbit/murine monoclonal blend
Contains a blend of (?) is a murine monoclonal IgM antibody
rabbit polyclonal antihuman IgG and Anti-C3d
Contains anti-IgG with no anticomplement activity
Anti-IgG (Rabbit polyclonal)
Murine monoclonal IgM antibody secreted by a hybridoma cell line
Anti-IgG (Gamma-done AHG)
The main component of this reagent is a murine monoclonal antibody to C3d.
Anti-C3d
will cause the agglutination of red blood cells coated with human C3d and/or C3b complement components.
Anti-C3d
- Involves injecting human serum or purified globulin into laboratory animals such as rabbits.
Classic method of AHG production
The (?) behaves as foreign antigen, the rabbit’s immune response is triggered, and an antibody to (?) is produced.
human globulin
human globulin
- Human IgG injected into a rabbit- results in
anti-IgG production
- Human complement components injected into a rabbit result in
anticomplement
PREPARATION OF AHG
This type of response produces a
polyclonal antiglobulin serum
can be made using polyclonal or monoclonal antibodies.
Polyspecific AHG
The two types of antibody production processes are very different from one another, yielding two very different advantages and disadvantages in their usage.
Polyspecific AHG
is usually prepared in rabbits.
Polyclonal AHG
are produced by immunizing one colony of rabbits with human immunoglobulin (IgG) antigen and another colony with human C3 antigen.
Conventional polyspecific antiglobulin reagents
Because of the heterogeneity of IgG molecules, using serum from many donors to prepare the pooled IgG antigen to immunize the rabbits and the pooling of anti-IgG from many immunized rabbits is essential in producing polyclonal reagents for routine use that are capable of detecting the many different IgG antibodies.
Polyclonal AHG
This is an advantage of using anti-IgG of polyclonal origin for antiglobulin serum.
Polyclonal AHG
allows for a broader spectrum of reactivity, but the source of antibody is limited to the life span of the inoculated animal.
Preparation of polyclonal AHG reagents
Pooled donor antigen
may be manufactured by combining polyclonal anti-IgG with either polyclonal or monoclonal anticomplement components.
Polyspecific antihuman globulin
Monoclonal antibody technique:
Kohler and Milstein
Monoclonal antibody technique:
Kohler and Milstein
Monoclonal antibody technique:
Kohler and Milstein
Useful in producing high-titer antibodies with well-defined specificities to IgG and to the fragments of C3
Monoclonal AHG Production
Monoclonal AHG Production
Procedure
1. Begins with the immunization of laboratory animals usually mice with [?]
2. After a suitable immune response, mouse spleen cells containing antibodysecreting lymphocytes are fused with [?].
3. The resulting hybridomas (?) are screened for anti -bodies with the required specificity and affinity.
4. The antibody- secreting clones may then be propagated in tissue culture or by inoculation into mice, in which case the antibody is collected as [?] (fluid accumulation in the abdomen of the mouse).
purified human globulin
myeloma cells
lymphocyte–myeloma hybrid cells
ascites
Because the clonal line produces a single antibody, there is no need for absorption to remove [?].
heterospecific antibodies
is a hybrid cell that is used as the basis for the production of large amounts of antibodies for therapeutic, research, or diagnostic applications.
hybridoma
The production of antibody is longer lasting than the polyclonal source, as the hybridoma can live indefinitely.
monoclonal AHG reagents
A monoclonal blend may be manufactured by blending
monoclonal anti-C3b, monoclonal anti-C3d, and monoclonal anti-IgG
can be manufactured by conventional or hybridoma technology
Monospecific antihuman globulin reagents
process is similar to that described above for polyspecific AHG
Monospecific AHG production
it contains only one antibody specificity
Monospecific AHG production
is produced as a monoclonal, polyclonal, or blended formula.
Monospecific anti-IgG
is produced as a monoclonal, polyclonal, or blended formula.
Monospecific anti-IgG
is produced as a monoclonal, polyclonal, or blended formula.
Monospecific anti-IgG
ANTIHUMAN GLOBULIN REAGENTS
- Polyspecific AHG
- Monospecific AHG
- Polyspecific AHG
a. Polyclonal AHG Production
b. Monoclonal AHG Production
ANTIBODIES REQUIRED IN AHG
- Anti-IgG
- Anticomplement
AHG must contain antibody activity to non-agglutinating blood group antibodies.
The majority of these antibodies are a mixture of IgG1 and IgG3 subclasses.
Rarely, non-agglutinating IgM antibodies may be found; however, they have been shown to fix complement and may be detected by anti-complement.
is the only FDA-licensed monoclonal IgG AHG in the United States
Gamma-clone anti-IgG (Immucor, Inc., Norcross, GA)
detects IgG1, IgG2, and IgG3, but as described in the package insert, it fails to react with IgG4.
Gammaclone
This is not regarded as a significant limitation since [?]- only antibody specificities are uncommon, have not been associated with acute hemolytic reactions, and often have high-titer low-avidity-type features associated with clinically insignificant reactivity.
IgG4
Some antibodies “fix” complement components to the RBC membrane after complexing of the antibody with its corresponding antigen.
Anticomplement
The terms most, some, and rare in the table refer to antibodies that bind complement the vast majority of the time, that show variability in their ability to bind complement, and that rarely bind complement, respectively.
Anticomplement
These membrane-bound complement components can be detected by the [?] activity in AHG.
Anticomplement
As a result of studies published during the 1960s indicating the need for [?] activity in AHG to allow the IAT to detect antibodies, a polyspecific reagent was introduced.
Anticomplement
Evidence was also presented showing that the presence of [?] activity would enhance the reactions of clinically significant antibodies (e.g., anti-Fya and anti-K).
Anticomplement
Antibodies Capable of Binding Complement MOST
ABO
Lea
Leb
Jka
Jkb
Sc1
Co3
Ge2
Ge3
Ii
P
P1PK
Vel
Antibodies Capable of Binding Complement SOME
Xga
LKE
Lan
Antibodies Capable of Binding Complement RARE
D
P1
Lua
Lub
Kell
Fya
Fyb
Coa
Cob
Dia
S
S
Yta
contains both anti-IgG activity and anti-C3d activity.
• Polyspecific AHG
• Because most clinically significant antibodies detected during antibody screening are IgG, the most important function of [?] is the detection of IgG antibodies.
polyspecific AHG
• Many blood banks have begun using [?] for routine pretransfusion testing, citing cost containment measures necessitated by the high number of repeats versus the rarity of complement-only detected antibodies such as anti-Jka.
monospecific anti-IgG
• Milam states that rare clinical transfusion intolerance, when using [?]over polyspecific AHG reagents to screen for unexpected antibodies and to test for blood group compatibility, offers reliability without interference from common and clinically insignificant IgMcomplement fixing antibodies.
monospecific anti-IgG
Antibody molecules and complement components are
globulins
Injecting an animal with [?] stimulates the animal to produce antibody to the foreign protein (i.e.,AHG).
human globulin
Serologic tests employ a variety of AHG reagents reactive with various human globulins, including anti-IgG antibody to the C3d component of human complement, and polyspecific reagents that contain both [?] activity.
antiIgG and anti-C3d
AHG reacts with [?] molecules, either bound to RBCs or free in serum.
human globulin
Washed RBCs coated with human globulin are agglutinated by [?].
AHG
detects in vivo sensitization of RBCs with IgG or complement components
direct antiglobulin test (DAT)
Clinical conditions that can result in in vivo coating of RBCs with antibody or complement are the following:
- Hemolytic disease of the fetus and newborn (HDFN)
- Hemolytic transfusion reaction (HTR)
- Autoimmune and drug-induced hemolytic anemia (AIHA)
o Maternal antibody coating of fetal RBCs
- Hemolytic disease of the fetus and newborn (HDFN)
o Recipient antibody coating donor RBCs
- Hemolytic transfusion reaction (HTR)
o Autoantibody coating individual’s RBCs
- Autoimmune and drug-induced hemolytic anemia (AIHA)
DAT Panel
Initial DATs include testing one drop of a 3% to 5% suspension of washed RBCs with [?].
polyspecific (anti-IgG, anti-C3d) reagent
DAT Panel
Positive results are monitored by a DAT panel using [?] to determine the specific type of protein sensitizing the cell.
monospecific anti-IgG and anti-C3d
Anti-IgG +
Anti-C3d +
WAIHA
Mixed-type AIHA (cold and warm)
Anti-IgG +
Anti-C3d -
WAIHA
Anti-IgG -
Anti-C3d +
CAS; PCH; WAIHA
The DAT with [?] is helpful in classifying AIHAs.
monospecific antiglobulin reagents
Other procedures and studies are necessary to diagnose and characterize which form of [?] is present.
autoimmune disease
CAS =
cold agglutinin syndrome
PCH =
paroxysmal cold hemoglobinuria
WAIHA =
warm autoimmune hemolytic anemia
states that “a positive DAT result alone is not diagnostic of hemolytic anemia.
AABB Technical Manual
Understanding the significance of this positive result requires knowledge of the [?].”
patient’s diagnosis; recent drug, pregnancy, transfusion, and hematopoietic transplantation history; and the presence of acquired or unexplained hemolytic anemia
Answering the following questions before investigating a positive DAT for patients other than neonates will help determine what further testing is appropriate:
1. Is there evidence of [?]?
2. Has the patient been [?] recently? If so, did the patient receive blood products or components containing ABOincompatible plasma?
3. Does the patient’s serum contain [?]?
4. Is the patient receiving any [?]?
5. Is the patient receiving [?]?
6. Is the patient receiving [?]?
7. Has the patient received a [?]?
in vivo hemolysis
transfused
unexpected antibodies
drugs
antilymphocyte globulin or antithymocyte globulin
intravenous immune globulin (IVIG) or intravenous Rh immune globulin (IV RhIG)
marrow or other organ transplant
The [?] is performed to determine in vitro sensitization of RBCs
INDIRECT ANTIGLOBULIN TEST
INDIRECT ANTIGLOBULIN TEST is used in the following situations:
a. Detection of [?] to potential donor RBCs (compatibility testing) or to screening cells (antibody screen) in serum
b. Determination of [?] using known antisera (e.g., weak D, any other antigen testing that requires IAT)
c. Titration of [?]
incomplete (nonagglutinating) antibodies
RBC phenotype
incomplete antibodies
[?], with sensitization occurring in vitro, looking for unknown antibody in the patient’s serum/plasma.
Indirect AHG (IAT)
(Note: If the [?] was identifying RBC antigens, the RBCs would be from the patient and the antibody would be the known reagent).
Indirect AHG (IAT)
[?] with sensitization already occurring in vivo.
Direct AHG (DAT)
(Note: The polyspecific AHG reagent is a mixture of anti-IgG and anti-C3d.)
Direct AHG (DAT)
Detects IgG and complement coated red cells
Direct Antiglobulin Test
Detects IgG and complement coated red cells
Indirect Antiglobulin Test
IgG attachment to red cells has occurred within the patient’s body One stage procedure
Direct Antiglobulin Test
IgG attachment to red cells has occurred during the incubation step
Indirect Antiglobulin Test
One stage procedure
Direct Antiglobulin Test
Two-stage procedure
Indirect Antiglobulin Test
Patient’s red cells are tested with antiglobulin reagent without an incubation step
Direct Antiglobulin Test
Test requires an incubation step before the addition antiglobulin reagent
Indirect Antiglobulin Test
Test for certain clinical conditions: HDFN, HTR, AIHA
Direct Antiglobulin Test
Used as a reaction phase of several tests immunohematology
Indirect Antiglobulin Test
