Transfusion - Module 1, Part 2

What does pepsin digestion produce?

2 Fc Fragments and 1 F(ab)2 Fragment.

Can the F(ab)2 fragment cause visible agglutination?

Yes, it can combine with Ag and may result in visible agglutination.

What is another name for 2-Mercaptoethanol (2ME)?

"Thiol" reagents.

What does 2-Mercaptoethanol (2ME) do?

Splits the Fab fragment by disrupting bonds between the H & L chain of an IgM molecule only.

What chain is disrupted by 2-Mercaptoethanol (2ME) in IgM?

The J (joining)-chain.

What does 2-Mercaptoethanol (2ME) create?

A free light chain and a fragment called "Fd".

What is 2-Mercaptoethanol (2ME) used for?

Separating mixtures of different classes of antibodies (IgM and IgG).

What fraction of antibodies of any specificity contain kappa light chains?

~2/3.

What fraction of antibodies of any specificity contain lambda light chains?

~1/3.

What are antibodies called if they are derived from many clones and react with a broad range of antigenic determinants?

Polyclonal.

What are antibodies called if they are derived from a single clone of antibody producing cells?

Monoclonal.

What disease states are monoclonal antibodies associated with?

Multiple myeloma, cold agglutinin disease, and autoimmune hemolytic anemia.

What determines the class of an immunoglobulin?

The heavy chain.

What are the five classes of immunoglobulins?

IgG (gamma), IgA (alpha), IgM (mu), IgD (delta), IgE (epsilon).

What is the molecular weight of IgG?

~150,000 daltons.

What percentage of total Ig is IgG?

~80%.

How many antigen binding sites does IgG have?

2.

Why is IgG called "incomplete"?

It cannot cause agglutination of red cells in saline at room temperature.

At what temperature does IgG react best?

37°C ("warm").

Why is IgG called "red cell immune"?

It is produced in response to foreign RBC Ag through transfusion or pregnancy.

How many subclasses of IgG are there?

4 (IgG 1,2,3,4).

Which IgG subclass does NOT activate complement?

IgG4.

Which immunoglobulin class is the ONLY one that can cross the placenta?

IgG.

In which immune response is IgG the main immunoglobulin?

Secondary response (formed late in primary).

What two conditions can IgG cause?

Hemolytic Disease of the Newborn (HDN) and hemolytic transfusion reactions (HTRXN).

What is the molecular weight of IgM?

~900,000 daltons.

What percentage of total Ig is IgM?

5-10%.

How many antigen binding sites does IgM have?

10 (pentamer with J-ring).

Why is IgM called "complete"?

It is able to cause visible agglutination of red cells in saline at room temperature.

Why is IgM called "naturally occurring"?

It is usually "non-immune" or naturally occurring (may be RBC stimulated).

In which immune response is IgM formed?

Primary response (small amounts in secondary).

Can IgM cross the placenta?

No, therefore it does not cause HDN.

Can IgM activate complement?

Yes, so it may cause hemolytic transfusion reactions.

What is the structure of secretory IgA?

A dimer joined by a J ring.

What protects secretory IgA against enzyme digestion?

The secretory piece.

What percentage of total Ig is IgA?

10-15%.

What severe reaction can occur in IgA deficient individuals receiving blood products?

Anaphylactic reaction due to anti-IgA.

What is the function of IgD?

Acts as an antigen receptor on B cells.

What is another name for IgE?

Allergic reaginic antibody.

What is IgE fixed to?

Tissue basophils.

What conditions does IgE play a major role in?

Allergies and asthma.

What does the DAT test detect?

In vivo sensitization of red cells.

What four conditions is the DAT useful for diagnosing/investigating?

Diagnosis of HDN, diagnosis of AIHA, investigation of drug sensitization of red cells, investigation of a transfusion reaction.

What control cells are used for the DAT?

IgG sensitized control cells (CC cells) and complement sensitized cells.

In the DAT procedure, what is the incubation step if the test is negative?

Incubate at R.T. for 5-10 min (allows detection of complement).

What does agglutination in the DAT indicate?

The cells have been coated with IgG and/or complement components in vivo.

What must be added to all negative AHG tests (DAT or IAT) as a control?

One drop of IgG sensitized red cells (CC cells).

What agglutination grade must be observed for the control to be valid?

1+ to 2+.

What three things does the CC control tell you?

AHG reagent was added, AHG reagent was active, and cells were washed adequately.

What is the sedimentation constant for IgG?

7S.

What is the sedimentation constant for IgM?

19S.

What is the half-life of IgG?

21 days.

What is the half-life of IgM?

5 days.

What is the half-life of IgA?

6 days.

Are Ag-Ab reactions specific, weak, and reversible?

Yes.

Which regions of the antibody are primarily concerned with antigen binding?

The variable regions.

Are Ag/Ab reactions covalent or non-covalent?

Non-covalent.

What are the four types of bonding involved in Ag/Ab reactions?

Hydrophobic, Hydrogen Bonds, Van der Waal's Forces, Electrostatic Forces.

Which type of bonding is considered the "major" bond and involves the exclusion of water molecules?

Hydrophobic.

What is another name for Van der Waal's forces due to the requirement of complementary shapes?

"Lock & Key" or "Closeness of fit".

What is another name for Electrostatic forces?

Coulombic Bonds.

What are the overall charges of Ag and Ab?

Ag is overall negative; Ab is overall positive.

What do all these forces determine?

The AVIDITY (strength) of antigen/antibody reactions.

What is the difference between affinity and avidity?

Affinity is the strength of a single Ag/Ab interaction; Avidity is the strength of all interactions combined.

What are the five types of antigen-antibody reactions?

Sensitization/Agglutination, Complement Fixation, Hemolysis, Neutralization/Inhibition, Precipitation.

Which types of Ag-Ab reactions are most common and applied in transfusion?

1, 2, and 3 (Sensitization/Agglutination, Complement Fixation, Hemolysis). 1-4 are applied in transfusion.

What happens during the first stage of sensitization/agglutination (Sensitization)?

Ab attaches, agglutination has not occurred (IgG) or complexes are too small to see.

What happens during the second stage of sensitization/agglutination (Lattice Formation)?

Cross-linking of Ag/Ab complexes to form visible agglutinates.

Which antibody class easily agglutinates RBCs?

IgM.

Which antibody class rarely agglutinates red cells without enhancement?

IgG.

In complement fixation, what components attach to the red cell?

Complement components up to C3.

Does hemolysis occur during complement fixation?

No.

How is complement fixation detected?

By the addition of an AHG reagent containing anti-C3.

How many complement components must be activated for hemolysis to occur?

All 9.

What is required for complement activation?

2 adjacent Fc fragments.

Which antibody class is a good complement activator?

IgM.

Which antibody class is a poor complement activator?

IgG.

In neutralization/inhibition, what type of antigen reacts with the antibody?

Soluble antigen.

In the neutralization/inhibition example with soluble Lea Ag, what does agglutination of the indicator cell mean?

Soluble Lea Ag is NOT present (no inhibition).

In the neutralization/inhibition example, what does NO agglutination of the indicator cell mean?

Soluble Lea Ag IS present (inhibition).

What class of antibodies is neutralization/inhibition used to detect?

IgM.

What type of antigen-antibody reaction forms a fine granular precipitate?

Precipitation.

What class of antibodies is usually involved in precipitation?

IgG (but not blood group Abs).

What is the optimum temperature for IgG antibodies?

37°C +/- 1°C.

What is the optimum temperature for IgM antibodies?

4°C.

How does decreased ionic strength affect Ag/Ab reactions?

It allows increased Ag/Ab reactions.

What is the "dosage" effect?

Some antibodies react stronger with homozygous rather than heterozygous antigen expression.

What is the normal pH of human plasma for Ag/Ab reactions?

6.5 - 7.5.

What is prozone?

A false negative due to excess antibody (all Ag sites saturated).

What is postzone?

A false negative due to excess antigen.

How long can test plasma/serum be stored before testing?

Generally <96 hrs (separated from cells).

Why is centrifugation used in Ag/Ab testing?

To move red cells closer together and overcome natural repulsion (zeta potential).

What happens if you under-centrifuge in Ag/Ab testing?

False negative (Ag/Ab reaction may not occur).

What happens if you over-centrifuge in Ag/Ab testing?

False positive (packs cells too tightly) OR false negative (if you re-suspend too hard).

What is the most common cause of transfusion-related deaths?

CLERICAL ERRORS.

What is rouleaux?

Cells arranged 'like stacks of coins' (not an Ag/Ab reaction).

What conditions cause rouleaux?

Abnormal proteins, reversed A/G ratio, plasma expanders (dextran, Ringers), Multiple Myeloma.

How is rouleaux resolved?

Saline Replacement.

What keeps RBCs apart in suspension?

Negative charges (~25 nm apart) from sialic acid residues.

How far can an IgM molecule span?

35 nm (allows visible agglutination).