Chapter 2 - Immunology: Basic Principles and Applications in the Blood Bank

Review chapter objectives

page 22 (34 online)

Antigens

molecules that bind to Abs or T-cell receptors

Allogenic antigens

nonself antigens (come from same species)

Autologous antigens

self antigens

Haptens

partial Ags that require a carrier molecule to elicit an immune response (medicine)

Ags contain different __ that can elicit different types of Abs

antigenic determinants (epitopes)

what reactions are most common in transfusion medicine

humoral (involve B cells)

plasma cells

B cells that produce majority of antibodies

memory B cells

B cells that respond rapidly to next exposure and transform into plasma cells

Immunogenicity

the bodies response to an Ag

factors that contribute to immunogenicity

chemical composition of Ag

degree of foreignness

size

dosage and Ag intensity

route of administration

Antibodies are ___ made up of __ polypeptide chains joined by a __ bond

glycoproteins, 4, disulfide

Ab light vs heavy chain

2 heavy chains (G,M,A,E,D)

2 lights chains (2 kappa or 2 lambda)

Ab constant region (heavy chain)

determines Ab class/isotype (IgG, IgM, IgA, IgE, IgD)

Ab variable region (light and heavy chains)

binds the antigen

Fab vs Fc

Fab = fragment antigen binding

Fc = fragment crystallizable (macrophage/complement binding region)

What Ig are most significant in immunohematology

IgG and IgM

IgM vs IgG

Mu vs. Gamma

both kappa or lambda

J chain vs no J chain

*10 vs 2 valence

10 vs 70-75 total serum %

5 vs 23 serum half life

*doesn’t vs does cross placenta

efficient for complement activation vs not as efficient

intravascular clearance of RBCs vs extravascular

immediate spin vs antiglobulin detection tests

Primary Immune Response

elicited on 1st exposure to Ag

lag phase of 5-10 days

IgM antibodies are produced first, followed by IgG

less antibody is produced

Secondary immune response

elicited on 2nd exposure to the same antigen

occurs within 1-3 days

mostly IgG antibodies, with lesser amounts of IgM antibodies

Ab levels are high and sustained for a longer amount of time

Affinity vs Avidity definition

Affinity = strength of the binding btw a single Ab and an epitope of an Ag

Avidity = overall strength of a reaction between several epitopes and antibodies

Affinity maturation

affinity increases after each exposure

high vs low affinity and binding speed

high = binds quickly

low = binds weakly

An ___ occurs when the Ag and Ab combine

immune complex

Strength of Ab-Ag binding is influenced by what Ag characteristics

size, shape, and charge

What holds immune complexes together

noncovalent forces and contribute to the avidity (strength) of the attachment

Noncovalent forces that effect Ab/Ag binding (4)

1. electrostatic forces (ionic) = attraction due to opposite charges

2. hydrogen bonding = attraction of two negatively charges groups (X-) for an H+ atom (ex = H2))

3. hydrophobic bonding = weak bonds formed as a result of the exclusion of water from the Ag/Ab complex

4. van der Waals forces = attraction between the electron cloud (-) of one atom and the protons (+) within the nucleus of another atom

cumulative effect contributes to Ag/Ab binding

RBC Antigens

located on RBCs as part of the cell membrane

glycoproteins or glycolipids

agglutinate with Abs

some Ags can elicit a stronger immune response than others (*D antigen) (Rh ±)

over 300 different blood group Ags, 36 different blood groups

RBC Antibodies

molecules in plasma (unclotted) or serum (clotted)

IgG antibodies are clinically significant; react at 37 C

IgM antibodies react at room temp or blow and are not usually considered significant, unless they activate complement (ABO antibodies)

Serum

liquid portion of blood leftover following clotting

Plasma

liquid portion of blood following collection with an anticoagulant

In vivo vs In vitro

vivo = body

vitro = laboratory (tube)

In Vivo Reactions

exposure to foreign antigens during transfusion or pregnancy may cause sensitization, resulting in production of alloantibodies (Abs other than self)

what test will detect alloantibodies

antibody screen test

detecting alloantibodies before transfusion helps prevent what

formation of Abs in vivo

Antibodies may activate complement proteins which can cause ___ destruction

RBC

Complement

complement proteins enhance the immunologic process when activated

final phases of complement activation cause the formation of a membrane attack complex (MAC), which lyses cells

2 complement pathways

1. classical pathway = activated by antibodies

2. alternative pathway = activated by foreign cell surface constituents (e.g., bacteria)

complement classical pathway steps

1. Ag/Ab complex exposes binding sire for C2 in Fc position

2. C1 activation results in activation for C2, C4, & C3

3. C proteins split into a & b (a=smaller, b=bigger)

4. C4bC2a aka C3 convertase converts C3 to active form (split into a/b)

5. C4bC2aC3b aka C5 convertase splits C5 to activate it

6. C5-C9 bind to RBC to form MAC

4 effects mediated by complement proteins

1. opsonization…clear immune complexes, enhance phagocytosis, promote release of enzymes from neutrophils

2. anaphylaxis…increase smooth muscle contraction and inflammation

3. lysis…kill foreign Ags by membrane lysis

4. chemotactic…recruit platelets and phagocytes

how are Ag/Ab reactions that occur in vitro (tube) detected

visible agglutination

agglutination = clumping of particles, often Ab/Ag

What do negative Ag/Ab in vitro reactions indicate

indicate a lack of agglutination

what are the 2 steps involved in agglutination

1. sensitization = Ab binds to Ag, but no visible agglutination occurs

2. lattice formation = Ab-coated cells cross-link to form visible agglutination

Factors that affect agglutination (sensitization vs lattice formation stage)

sensitization

• temperature…IgG 37 C, IgM < 22 C

• incubation time…immediate-spin after a specific time at 1-8 C, room temperature (20 c), or 37 C

• pH…7.0 (physiological is ideal)

• Ionic strength…can be adjusted with reagents

lattice formation

• zeta potential…distance between cells caused by charged ions

• zone of equivalence…Ag/Ab concentrations (# of binding sites of a multivalent Ag/Ab are equal)

• centrifugation…time and speed of centrifugation to bring cells close together

zone of equivalance…prozone vs postone

prozone = excess Ab

postzone = excess Ag

Grading agglutinatin reactions

4+ = red cell button is a solid agglutinate, clear background

3+ = several large agglutinates; clear background

2+ = many medium-sized agglutinates; clear background

1+ = medium and small sized agglutinates; background is turbid with many free red cells

0 = no agglutinated RBCs are visible; red cells are observed flowing off the red cell button during the process of grading

In addition to agglutination, what can also indicate an Ag/Ab reaction

hemolysis

Hemolysis is normally caused by activation of what

complement

RBC button if hemolysis occurs

smaller and a pink-red supernatant is observed after centrifugation

should fresh, nonhemolyzed samples be used for testing

YES

What prevents complement acitvation in vitro by chelating calcium

anticoagulants

Immunohematology ___ are used to detect Ag/Ab reactions

reagents

Ab/Ag reactions in vitro appear as…

agglutination or hemolysis

testing methods for Ag/Ab reactions

tube testing, gel technology, solid-phase adherence technology

all routine test methods use a source of Ag/Ab to detect…

agglutination or hemolysis

routine testing, sources of Ag

1. reagent RBCs…commercially prepared, known source of Ag

2. patient or donor RBCs…usually an unknown source of Ag, RBCs are tested with commercial antisera to determine antigen identity

routine testing, sources of Ab

1. reagent antisera

   1. commercially prepared

   2. known source of Ab

2. patient or donor serum or plasma

   1. usually unknown

   2. serum or plasma is tested with commercial RBCs to determine identity of antibody or antibodies

5 examples of routine testing procedures done in immunohematology lab

1. ABO/D typing (forward typing)

2. ABO serum testing (reverse grouping)

3. Antibody screen

4. Antibody identification

5. Crossmatch

ABO/D typing (forward grouping) purpose, Ag source, and Ab source

Purpose = detect A, B, and D antigens

Ag source = Patient’s RBCs

Ab source = commercial anti-A, anti-B, and anti-D

ABO serum testing (reverse grouping) purpose, Ag source, and Ab source

purpose = detects ABO antibodies

Ag source = reverse grouping cells (A1 and B)

Ab source = patient’s serum or plasma

Antibody screen purpose, Ag source, and Ab source

purpose = detects Abs with specificity to red cell antigens

Ag source = screening cells

Ab source = patient’s serum or plasma

Antibody identification purpose, Ag source, and Ab source

purpose = identifies specificity of red cell antibodies

Ag source = panel cells

Ab source = patient’s plasma or serum

Cross match purpose, Ag source, and Ab source

purpose = determines serologic compatibility between donor and patient before transfusion

Ag source = Donor’s red cells

Ab source = patient’s serum or plasma