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ANTIGLOBULIN TESTING Flashcards

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1
Q

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).

A

ANTIHUMAN GLOBULIN TEST

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2
Q

It detect IgG or complement-sensitized RBCs?

A

ANTIHUMAN GLOBULIN TEST

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3
Q

What is the 2 MAJOR TYPES OF BLOOD GROUP ANTIBODIES

A

IgM, IgG

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4
Q

Bind to corresponding antigen and directly agglutinate RBCs suspended in saline

A

IgM

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5
Q

It is a pentamer, and is considered to be the best agglutinin

A

IgM

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6
Q

“nonagglutinating” or “incomplete antibodies” – because their single monomer structure is too small to directly agglutinate sensitized RBCs

A

IgG

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7
Q

precipitin so it produces small clumps as compared to agglutination.

A

IgG

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8
Q

1945, they described the use of the antiglobulin test for the detection of weak and nonagglutinating Rh antibodies in serum.

A

Coombs, Mourant, and Race

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9
Q

1946 ,Coombs and coworkers described the use of ?

A

AHG to detect in vivo sensitization of the RBCs of babies suffering from HDFN.

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10
Q

In 1908, Moreschi described the principle of the

A

Anti-globulin test

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11
Q

In 1908 - Moreschi described the principle of the Anti-globulin test which included what blood group antibodies and associated antigen.

A

Kell blood group antibodies and associated antigen

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12
Q

Injection of human serum into rabbits to produce antihuman serum

A

Coombs procedure

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13
Q

In Coombs procedure the one we see in this procedure is?

A

Hemagglutination

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14
Q

Result of RBCs has been sensitized by an antibody

A

Hemagglutination

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15
Q

In 1947 - Coombs and Mourant demonstrated that the antibody activity that detected

A

Rh

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16
Q

Coombs and Mourant demonstrated that the antibody activity that detected Rh
o antibodies were associated with the ?

A

anti-gamma globulin fraction

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17
Q

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.

A

Dacie

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18
Q

Warm reacting antibodies

A

IgG

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19
Q

Cold reacting antibodies

A

IgM

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20
Q

The Antiglobulin test can be used to detect RBCs sensitized with?

A

IgG alloantibodies, IgG autoantibodies, and complement components.

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21
Q

Sensitization can occur either in?

A

Vivo and vitro

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22
Q

Ag-Ab binding happened inside the body? In vivo or in vitro?

A

vivo

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23
Q

outside the body? In vivo or in vitro?

A

vitro

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24
Q

is detected by a one stage procedure because their already sensitized RBC in body.

A

vivo sensitization

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25
vivo sensitization is detected by?
Direct antiglobulin test (DAT)
26
The use of AHG to detect in (?) sensitization of RBCs is a two-stage technique.
vitro sensitization
27
vitro sensitization is detected by
Indirect antiglobulin test (IAT)
28
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.
29
Defined by FDA and CBER (Center for Biologics Evaluation and Research)
ANTIHUMAN GLOBULIN REAGENTS
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FDA and CBER transcribe?
Food and Drug Administration, Center for Biologics Evaluation and Research
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ANTIHUMAN GLOBULIN REAGENTS 2 types?
POLYSPECIFIC, MONOSPECIFIC
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is the one we check because it presents in all pathway.
- C3
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Antibody to human IgG and to the C3d component of the human complement, contains more than 1 type of antibody.
POLYSPECIFIC
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POLYSPECIFIC commonly used?
Anti-C3b, anti- C4b and anti-C4d may also be present
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- contain only one antibody specificity: either anti IgG or antibody to specific complement components (C3b or C3d)
MONOSPECIFIC
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Monospecific commonly used?
: anti-IgG and anti-C3b, anti-C3d
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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
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Anticomplement reagents?
Anti-C3b and anti-C3d reagents
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Often a blend of monoclonal anti-C3b and monoclonal C3d
- Anticomplement
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Complement is the start of the reaction that is common in 3 pathways
classical, alternative, mannose)
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foreign antigen to the rabbits
- Human globulin
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- Many Ag to bind
POLYCLONAL ANTIBODIES
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- Mixture of antibodies from different plasma cell clones. - Directed against multiple types of epitopes.
POLYCLONAL ANTIBODIES
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determinant site that is found in the antigen to be able to recognize by the antibody
o Epitopes
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- 1 Ag can only be bind. - Derived from one clone of plasma cells and recognize a single epitope.
MONOCLONAL ANTIBODIES
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can be used to produce monoclonal antiglobulin serum
- Hybridoma technology
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POLYCLONAL AHG PRODUCTION usually prepared using?
rabbits
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IN POLYCLONAL AHG PRODUCTION, Large volumes of antibody are required (?) and (?) may be used
Sheep or Goats
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- If the rabbit is injected with a complement, it will produce
Monospecific Polyclonal Anti-C3b, Anti-C3d.
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- If the rabbit is injected with an IgG, it will produce
Monospecific Polyclonal Anti-IgG.
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When we mixed both Monospecific Polyclonal Anti-C3b, Anti-C3d and Monospecific Polyclonal Anti-IgG together, it can produce
Antihuman Globulin Polyspecific Polyclonal Blend
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means there is only 1 Ab but can detect many epitopes (reacts)
Monospecific Polyclonal
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- Recognize multiple epitopes on any one antigen.
POLYCLONAL ANTIBODIES
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contains a heterogenous complex mixture of antibodies of different affinity.
- Serum obtained
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made up mainly of IgG subclass. (4 subclasses of IgG)
- Polyclonals
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- often used to generate polyclonal antibodies that target unique epitopes, especially for protein families of high homology
- Peptide immunogens
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MONOCLONAL AHG PRODUCTION also called?
hybridoma technology
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MONOCLONAL AHG PRODUCTION was discovered by?
Kohler and Milstein
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in monoclona AHG production, Immunization of laboratory animals usually use is (?) with purified human globulin.
mice
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For HYBRIDOMA to form we need to incubate it to
HAT medium (hypoxanthine–aminopterin–thymidine)
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IN PREPARING ANTI COMPLEMENT in MONOCLONAL AHG PRODUCTION we will have?
Monoclonal anti-C3b, d, or g (1 only Ab target)
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IN PREPARING ANTI-IGG in MONOCLONAL AHG PRODUCTION we will have?
Monoclonal ant-IgG (1 only Ab target)
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If we mix the anti-C3b,d,g and anti-IgG, we will have an
anti-human globulin (AHG) polyspecific monoclonal blend
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- Contains only one antibody specificity
MONOSPECIFIC AHG
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is produced as a monoclonal, polyclonal or blended formula.
- Monospecific anti-IgG
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* Anti-IgG: Majority are mixture of (?) and (?) subclasses
IgG1 and IgG3
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- High specificity - Detects only one epitope on the antigen. - Consist of only one antibody subtype (e.g. IgG1, IgG2, IgG3, iGG4)
MONOCLONAL ANTIBODIES
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- Antibody molecules and complement components are
globulins
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reacts with human globulin molecules, either bound to RBCs or free in serum. Washed RBCs coated with human globulin are agglutinated.
AHG
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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
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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
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RBC have metabolites of the drugs attached which usually cause by ?
ibuprofen
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o One drop of 3% to 5% suspension of washed RBCs with polyspecific (anti-IgG, anti-C3d) reagents.
- DAT Panel
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Anti-IgG (+) and Anti-C3d (+)
67% of Warm Autoimmune Hemolytic Anemia
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Anti-IgG (+) and Anti-C3d (-)
20% Warm Autoimmune Hemolytic Anemia cases
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Anti-IgG (-) and Anti-C3d (+)
(13%) of Cold Hemagglutinin disease (CHD), Paroxysmal Cold Hemoglobinuria (PCH), Warm Autoimmune Hemolytic Anemia (WAIHA)
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In vivo sensitization: Maternal Ab coting fetal RBC
HDN
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In vivo sensitization: Recipient Ab coating donor RBC
HTR
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In vivo sensitization: Autoantibody coting individual RBC
AIHA
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In Hemolytic Disease of the Newborn (HDN), what we check for DAT will be
Fetal RBC
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In Hemolytic Transfusion Reaction (HTR) we will be checking the
Recipient blood
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- Own antibodies are attacking its own cell
Autoantibody
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- performed to determine in vitro sensitization of RBCs and is used in the following situations
INDIRECT ANTIGLOBULIN TEST (IAT)
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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)
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Ab detection
copatibility testing, Ab screening
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Ab identification
Ab panel
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Ab titrataion
Rh Ab titer
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RBC phenotype
RBC Ag detection
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- Cells suspended in saline:
INCUBATION TIME
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incubation times vary between (?) if there is no reaction medium
30-120 minutes
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shortened the Incubation time to 10 to 15 minutes
Low-Ionic-Strength Solution, Polyethylene Glycol

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