Anti-Human Globulin Testing

Question 1

Antihuman Globulin test is also referred as ___

Answer 1

Coombs’ Test

Question 2

The principle of AHG is that antihuman globulins obtained from immunized nonhuman species bind to human globulins such as ___ or ___ , either free in serum or attached to antigens on red blood cells (RBCs).

Answer 2

IgG or Complement

Question 3

The AHG test is an essential testing methodology for __ medicine.

Answer 3

transfusion medicine

Question 4

2 major types of blood group antibodies

Answer 4

IgM and IgG

Question 5

Because of their large pentameric structure, _ antibodies bind to corresponding antigen and directly agglu- tinate RBCs suspended in saline

Answer 5

IgM

Question 6

Some _ antibodies are termed nonagglutinating, or incomplete antibodies, because their single monomer structure is too small to directly agglutinate sensitized RBCs

Answer 6

IgG

Question 7

Adding AHG reagent containing anti-IgG to RBCs sensitized with IgG antibodies allows for _ of these sensitized cells

Answer 7

hemagglutination

Question 8

Antiglobulin tests detect _ or complement-sensitized RBCs.

Answer 8

IgG

Question 9

2 types of AHG test

Answer 9

Direct Antiglobulin test – in vivo
Indirect Antiglobulin test – in vitro

Question 10

In 1946, Coombs and coworkers2 described the use of AHG to detect __ __ sensitization of the RBCs of neonates suffering from hemolytic disease of the fetus and newborn (HDFN)

Answer 10

in vivo

Question 11

The Coombs’ test involved the injection of human serum into rabbits to produce __ serum.

Answer 11

antihuman serum

Question 12

The antiglobulin test can be used to detect RBCs sensitized with IgG __ , IgG __, and complement components.

Answer 12

IgG alloantibodies, IgG autoantibodies

Question 13

The use of AHG to detect in vitro sensitization of RBCs is a two-stage technique referred to as the __.

Answer 13

indirect antiglobulin test (IAT)

Question 14

2 Reagents use in AHG

Answer 14

Polyspecific AHG
Monospecific AHG

Question 15

Regaent that contains anti-IgG and C3D

Answer 15

Polyspecific AHG

Question 16

Reagent that contains only anti-IgG

Answer 16

Monospecific AHG

Question 17

Principles of the Antiglobulin Test:
- Antibody molecules and complement components are __.

Answer 17

globulins

Question 18

AHG reacts with human globulin molecules, either __ or __.

Answer 18

bound to RBCs or free in serum

Question 19

The __ detects in vivo sensitization of RBCs with IgG or complement components.

Answer 19

direct antiglobulin test (DAT)

Question 20

Clinical conditions that can result in in vivo coating of RBCs with antibody or complement are the following:

Answer 20

• Hemolytic disease of the fetus and newborn (HDFN)
• Hemolytic transfusion reaction (HTR)
• Autoimmune and drug-induced hemolytic anemia (AIHA).

Question 21

Principle of Direct AHG

Answer 21

Washed Patient RBC (sentisized in vivo) + (coombs’ Sera) AHG Reagent Polyspecific = Visual Red Cell Agglutination

Question 22

___ is used to demonstrate in vitro reactions between red cells and coating antibodies, as in antibody detection, antibody identification, blood grouping, and compatibility testing.

Answer 22

Indirect Antiglobulin Test

Question 23

The IAT is performed to determine in vitro sensitization of RBCs and is used in the following situations:

Answer 23

• Detection of incomplete (nonagglutinating) antibodies to potential donor RBCs (compatibility testing) or to screen- ing cells (antibody screen) in serum
• Determination of RBC phenotype using known antisera (e.g., weak D, any other antigen testing that requires IAT)
• Titration of incomplete antibodies

Question 24

Factors Affecting the Antiglobulin Test

Answer 24

• Ratio of serum to cells
• Reaction medium
• Temperature
• Incubation time
• Washing of RBCs
• Saline for washing
• Addition of AHG
• Centrifugation for reading

Question 25

True or False. Increasing the ratio of serum to cells DECREASES the sensitivity of the test system.

Answer 25

False. Increasing the ratio of serum to cells INCREASES the sensitivity of the test system.

Question 26

Ratio of Serum to Cells

Answer 26

2 drops of serum and 1 drop of a 5% volume of solute per volume of solution (v/v) suspension of cells

Question 27

Factors Affecting the Antiglobulin Test: Reaction mediums include _, _ and _.

Answer 27

albumin, LISS, and polyethylene glycol.

Question 28

The macromolecules of _ allow antibody-coated cells to come into closer contact with each other so that agglutination occurs.

Answer 28

albumin

Question 29

In 1965, Stroup and MacIlroy42 reported on the increased _ of the IAT if albumin was incorporated into the reaction medium.

Answer 29

sensitivity

Question 30

The use of _ does not seem to provide any advantage over LISS techniques and adds to the COST of the test.

Answer 30

albumin

Question 31

_ enhance antibody uptake and allow incubation times to be decreased—from 30 to 60 minutes to 10 to 15 minutes—by reducing the zeta potential surrounding the RBC.

Answer 31

Low ionic strength solutions (LISS)

Question 32

(LISS) enhance antibody uptake and allow incubation times to be decreased—from 30 to 60 minutes to __

Answer 32

10 to 15 minutes

Question 33

In LISS, Increasing the serum-to-cell ratio increased the ionic strength of the reaction mixture, leading to a _ in sensitivity and counteracting the shortened incubation time of the test.

Answer 33

decrease

Question 34

_ is a water-soluble linear polymer and is used as an additive to increase antibody uptake.

Answer 34

Polyethylene glycol (PEG)

Question 35

_ is the AHG reagent of choice with PEG testing to avoid false-positive reactions.

Answer 35

Anti-IgG

Question 36

_ action is to remove water molecules surrounding the RBC (the water of hydration theory), thereby effectively concentrating antibody.

Answer 36

Polyethylene glycol (PEG)

Question 37

Majority of IgG antibodies is optimal at _

Answer 37

37˚c

Question 38

For cells suspended in saline, incubation times may vary between _ and _ minutes

Answer 38

30 and 120 minutes

Question 39

Incubation time: Significant antibodies can be detected after _ minutes

Answer 39

30

Question 40

When both the DAT and IAT are performed, RBCs must be saline-washed a minimum of _ times before adding AHG reagent

Answer 40

3

Question 41

Inadequate washing may result in a __ reaction because of neutralization of the AHG reagent by residual unbound serum globulins.

Answer 41

false-negative

Question 42

The _ phase of a DAT and IAT is one of the most important steps in testing.

Answer 42

washing

Question 43

True or False? Washing should be performed immediately after being removed from the incubator and in as short a time as possible to minimize the elution of low-affinity antibodies.

Answer 43

True

Question 44

Centrifugation at each wash should be sufficient to provide a _ cell button and therefore minimize the possible loss of cells with each discard of saline.

Answer 44

firm cell button

Question 45

Ideally, the saline used for washing should be _ (suggested open expiration of 30 days)

Answer 45

fresh

Question 46

Saline used for washing buffered to a pH of __ to __.

Answer 46

7.2 to 7.4.

Question 47

Saline stored for long periods in plastic containers has been shown to _ in pH, which may _ the rate of antibody elution during the washing process, yielding a false-negative result

Answer 47

decrease in pH increase rate of antibody

Question 48

Significant levels of bacterial contamination in saline have been reported; this situation can contribute to _ results.

Answer 48

false-positive

Question 49

Addition of AHG • Should be added _ after washing

Answer 49

immediately

Question 50

True or False. Centrifugation of the cell button for reading of hemagglutination along with the method used for resuspending the cells is a crucial step in the technique.

Answer 50

True

Question 51

The CBER- recommended method for the evaluation of AHG uses _ relative centrifugal forces (RCFs) for _ seconds

Answer 51

1,000 rcf, 20 seconds

Question 52

Inadequate resuspension may cause weak _ results

Answer 52

false-postive

Question 53

Too vigorous may yield _ results

Answer 53

false-negative