Chapter 2 Immunology Basics & Applications

Question 1

How is it possible to have an antibody to red cell antigens other than the ABO Blood group system?

Answer 1

1. Exposure through red cell transfusion
2. Exposure through pregnancy
   a) traumatic event
   b) at birth

Question 2

What are allogeneic antigens and autologous antigens?

Answer 2

Allogeneic antigens: non-self-antigens
Autologous antigens: self-antigens

Question 3

What factors contribute to immunogenicity regarding specifically dosage & antigen density?

Answer 3

Dosage and antigen density - The more red cells introduced and the more antigens that they carry the more likely there is to be an immune response.

Question 4

What are haptens?

Answer 4

Haptens are partial antigens that require a carrier molecule to elicit an immune response (e.g., medications)

Question 5

What is an alloantibody?

Answer 5

An alloantibody is an antibody to an antigen from the same species.

Question 6

What are antibodies made of?

Answer 6

Antibodies are glycoproteins made of 4 polypeptide chains joined by disulfide bonds

2 heavy chains
2 light chains (either kappa or lambda chains)

Constant region (heavy chains) determines antibody class
IgG, IgA, IgM, IgD, or IgE

Variable region (light and heavy chains) binds the antigen

Question 7

What type of immune response is most common in transfusion medicine?

Answer 7

Most reactions are humoral in transfusion medicine, i.e. they involve B lymphocytes.

Plasma cells: B cells that produce the majority of antibodies
Memory B cells: B cells that respond rapidly to next exposure and transform into plasma cells

Question 8

What is the preferred temperature for IgM versus IgG for agglutination?

Answer 8

IgM - 4 to 20C
IgG - 37C

Question 9

What antibody of the mothers can cross the placenta?

Answer 9

IgG can cross the placenta.

Question 10

Do IgG antibodies result in visible hemagglutination?

Answer 10

No. IgG antibodies are small. Even if they attach to red cells, they don’t cause visible hemagglutination. We must augment the reaction.

Question 11

What is the concern if IgG antibodies attach to red cells in vivo?

Answer 11

If IgG antibodies attach to the red cells they are usually removed from circulation in the spleen or liver (extravascularly).

Question 12

What antibody activates the classical complement pathway?

Answer 12

IgM antibodies can easily activate the classical complement pathway, due to its size and valency. If IgM antibodies are attached to the red cells it will result in intravascular lysing of thered cells

Question 13

What type of antibodies are the ABO blood group?

Answer 13

ABO blood group antibodies are typically IgM. We do room temperature testing for them. We are able to immediately spin the tubes and observe an immediate reaction if the antigens are present.

Question 14

What is the difference between the primary and secondary immune response?

Answer 14

Primary - Elicited on 1st exposure to antigen, lag of 5-10days, IgM antibodies are produced then IgG.
Secondary - anamnestic response, elicited on 2nd exposure to same antigen, w/in 1-3 days, mostly IgG antibodies (with lesser of IgM). Antibody levels are high and sustained for a longer amount of time.

Question 15

What do antigens and antibodies consist of?

Answer 15

Antigens are made of glycoproteins or glycolipids.
Note: Rh antigens are only proteins w/ NO carb or lipids.

Antibodies are made of glycoproteins.

Question 16

Where do you find antigens versus antibodies?

Answer 16

Antigens - located on RBCs; part of the cell membrane.
Antibodies - molecules in plasma or serum.

Question 17

What is the purpose of an antibody screen test?

Answer 17

An antibody screen test will detect alloantibodies

Detecting alloantibodies before transfusion helps prevent formation of Ag-Ab complexes in vivo which would lessen the survival of the transfused cells.

Antibodies may activate complement proteins, which can also cause RBC destruction

Question 18

Are Ag:Ab reactions reversible?

Answer 18

Yes, Binding of antibody (plasma) to antigen (red cell surface) forms an immune complex and is reversible

Question 19

What happens after performing a test at t37C and then you remove it from the waterbath and let it sit?

Answer 19

The antibody may dissociate from the antigen and no longer stay attached.

Question 20

What is affinity?

Answer 20

Goodness of fit
“lock and key”
Strength of binding between a single epitope and antibody is referred to as affinity
Held together by non-covalent forces such as:
Electrostatic forces, hydrogen bonding, and Van der Waals forces

Question 21

What is avidity?

Answer 21

Overall strength of several antigen-antibody reactions is referred to as avidity

Question 22

What are the 2 steps involved in agglutination?

Answer 22

There are 2 steps involved in agglutination:

Sensitization: antibody binds to antigen, but no visible agglutination occurs

Lattice formation: antibody-coated cells cross-link to form visible agglutination

Question 23

What are the factors affecting agglutination?

Answer 23

Factors affecting agglutination:
1. Sensitization
a) Temperature IgG 37C; IgM <=22C
b) Incubation time - immediate spin or after a specific time
c) pH - 7.0 (physiologic is ideal), by manf.
d) Ionic Strength
2. Lattice Formation
a) Zeta potential - distance between cells caused by charged ions
b) Zone of equivalence - Ag & Ab concentrations
c) Centrifugation - time & speed to bring cells closer together

Question 24

What is the problem with an isotonic environment in TS?

Answer 24

In an isotonic Na and Cl are attracted to opposite charges on Ab and Ag which hinders the reaction (BAD) but a reduced ionic environment can be used to improve amount of Ab uptake onto red cells (GOOD).

Question 25

What is zeta potential?

Answer 25

Zeta Potential:
Definition:
The force of repulsion between red cells in physiological saline solution
Red cells carry a negative charge and attract positive ions (cations) to create a cloud
The force between the cells at the plane of shear or slipping plane is the zeta potential

Question 26

Why can’t IgG cause visible agglutination?

Answer 26

IgM can span the space between cells as a result of zeta potential (Typically 25nm) and cause visible agglutination; IgG is too small and might not cause visible agglutination

Question 27

What is the optimal concentration of antibody to antigen called for lattice formation?

Answer 27

Optimal Concentrations of antibody (ab) and antigen (ag):
Agglutination when ag and ab fall within the zone of equivalence
This is what we want for our testing
This is why we use 2-5% red cell suspensions

Question 28

What are the zones of antibody excess and antigen excess called? What is the result in those zones?

Answer 28

Antibody excess = prozone or prezone
Antigen excess = postzone
False negatives if not in the zone of equivalence (no visible agglutination will occur)

Question 29

What is the purpose of centrifugation?

Answer 29

The purpose of the centrifuge is to mechanically bring the cells closer together to allow for interactions and agglutination.

Question 30

What causes false positives and/or false negatives in centrifugation if not done right?

Answer 30

Centrifugation effects:
Too long = false positive if cells are so packed together we interpret as agglutination
Too short = false negative – if we don’t allow them time to get together
Too fast = false positive if cells are so packed together we interpret as agglutination
Too slow = false negative – if they are not able to get together

Check package insert of reagent to determine optimal speed and time.

Question 31

How do you grade agglutination reactions in a tube?

Answer 31

Read by dislodging cell button by gently rocking tubes
You must allow the liquid in the tubes to flow down at least halfway to drag the cells with it. As you tip the tube back up, the liquid flows over the cells, helping to dislodge them.
If too vigorous you can break button down (was 3+ but shook down to 1+ or worse yet 1+ to a 0)
Negative reactions
Need to gently re-suspend all cells from the bottom of the tube
Recorded as “0” not “-”

Question 32

What is a 2+ agglutination reaction?

Answer 32

Many medium-sized agglutinates; clear background.

Question 33

Besides agglutination, what other physical sign can indicate Ag:Ab reaction?

Answer 33

In addition to agglutination, hemolysis can also indicate an antigen–antibody reaction

Hemolysis is normally caused by complement activation

The RBC button is smaller and a pink to red supernatant is observed after centrifugation

Question 34

Should hemolyzed samples be used for testing?

Answer 34

No.

Anticoagulants (like EDTA) prevent complement activation in vitro by chelating calcium
Complement cascade requires calcium
Will only see in serum tubes

Question 35

What are sources of antigens for routine testing?

Answer 35

1. Patient or donor RBCs
   - usually unknown antigens
   - RBCs are tested with commercial antisera to determine antigen identify
2. Reagent RBCs
   - commercially prepared
   - known source of antigen

Question 36

What are sources of antibody for routine testing?

Answer 36

1. Patient or donor serum or plasma
   - usually unknown
   - Serum or plasma is tested with commercial RBCs to determine Ab identify
2. Reagent antisera
   - commercially prepared
   - known source of antibody

Question 37

What does ABO/D typing - forward grouping (typing) look for?

Answer 37

ABO/D typing - forward grouping (typing)
Looks for “unknown” antigens on the patient cells.
Uses patient red cells (3-5%) – source of antigens
These are considered “unknown”
Uses known antisera – commercial reagent
Anti-A, Anti-B, and Anti-D

Hemagglutination means the antigen is present.
No agglutination means the antigen is not present.

Question 37

How is ABO/D typing - forward grouping performed?

Answer 37

ABO/D typing - forward grouping:
Tube test is adding one drop of specific antisera and one drop of patient 3-5%. Mix, immediate spin (3400rpm for 15 seconds) and read.

Question 38

What does ABO typing - reverse grouping do?

Answer 38

ABO typing - reverse grouping
Looks for “unknown” antibodies in the patient’s plasma.

This is to double-check the forward grouping

Uses known commercial red cells (antigen)
A cells (AC) and B cells (BC)
Uses patient Plasma – source of antibodies
These are considered “unknown”

Hemagglutination means the antibody is present.
No agglutination means the antibody is not present.

Question 39

How is ABO typing - reverse grouping performed?

Answer 39

Tube test is adding one drop of specific commercial cells and 2 drops of patient plasma. Mix, immediate spin, and read.

Question 40

What is an antibody screen?

Answer 40

The purpose of this test is to look for the presence of any unexpected (non-ABO), clinically significant antibodies
Uses commercial phenotyped red cells as source of known antigens (2 to 3 vials)
Uses patient plasma – source of Antibody

Question 41

Who is an antibody screen performed on?

Answer 41

An antibody screen is done on the following:
Allrecipients of blood or blood products
All blood or blood product donors
Expectant people

Question 42

What is done if the antibody screen is positive?

Answer 42

An investigation. A test is then done that uses a panel of commercial phenotyped red cells (10 to 20 vials) as the source of antigen (known) and the patient plasma as the source of antibody (unknown).

Question 43

What is phenotyping?

Answer 43

Phenotyping: If we identify the antibody and that individual requires a red cell transfusion, we must not give them blood that contains that antigen

In order to find out if the donor red cell unit has that antigen, we must type using a specific antisera.
For example, if a patient has Anti-Fya we must give them red cells that lack Fya.
We would make 3-5% of donor cells and add a drop to one drop of Anti-Fya.

Question 44

What is a crossmatch?

Answer 44

After a type and screen is done to rule out clinically significant antibodies the recipient’s plasma is combined with the donor’s red cells to see if they are compatible in a tube.

Question 45

How is a crossmatch done?

Answer 45

We make a 3-5% of the donor red cells (source of antigens) and add one drop of that into 2 drops of recipient’s plasma (source of antibodies)

If there is no agglutination in a tube, we feel it is safe to give that unit of blood to that patient.

Most of the time this is confirmation of ABO groups

(Note- gets more complicated if the recipient has a positive antibody screen)