Immunohematology

Comprehensive Review

Epitope (antigenic determinant)

Which term refers specifically to the smallest portion of an antigen that is recognized by an antibody?

Immunogens can elicit an immune response; antigens may not

What best distinguishes an immunogen from an antigen?

IgM

Which antibody is most efficient at activating complement?

IgG - reacts best at 37°C and requires AHG for detection.

A weak antibody is detected only at AHG phase. Which is most likely?

IgM can span larger distances between RBCs; Pentameric structure → better lattice formation → visible agglutination.

Why does IgM produce stronger agglutination than IgG in saline?

IgM

Which immunoglobulin predominates in a primary immune response?

Secondary

A patient previously exposed to an antigen shows a rapid, high-affinity response. This is what kind of response?

Higher affinity antibodies

Which is TRUE of secondary immune responses?

IgG or IgM bound to antigen

What initiates the classical complement pathway?

C5b–C9

Which complement component forms the Membrane Attack Complex (MAC)?

Cell lysis, Opsonization, Inflammation

What are 3 functions of complement?

Too much antibody = false negative

What happens in antibody excess (prozone)?

Reducing ionic cloud around RBCs

Low ionic strength solution (LISS) enhances agglutination by:

False positives

Overcentrifugation may result in:

4+

A reaction showing one solid clump with clear background is graded:

1+

Microscopic agglutination with many free cells is:

Antigens encoded by a single gene or gene cluster

In transfusion medicine, a blood group system is defined by:

What is “dosage”?

Stronger reaction with homozygous cells

Codominant

Which inheritance pattern applies to ABO blood groups?

A, B, AB, O

A mother is type A (AO), father is type B (BO). Possible child types?

O

If both parents are type O, what are the possible offspring?

91%

A patient has anti-K (K antigen frequency ≈ 9%). What % of units are compatible?

31%

K-negative = 0.91
Fya-negative = 0.34
0.91 × 0.34 = ~0.31 (31%)

Patient has anti-K (9%) and anti-Fya (66%). Probability of compatible units?

Direct exclusion

A child is type AB. Alleged father is type O. Conclusion?

Missing expected antigen

What indicates indirect exclusion?

Patient recently transfused

Molecular testing is most useful when:

Determines genotype independent of transfusion

What is a major advantage of molecular typing?

Antigen

Antigens bind antibodies, but may not trigger an immune response.
Binding ≠ stimulating

Which term refers to any substance capable of binding to an antibody?

Some antigens cannot stimulate an immune response

Immunogenicity requires immune activation, not just binding.
Hint: If it doesn’t “activate,” it’s not an immunogen

All immunogens are antigens, but not all antigens are immunogens because:

Epitope

Epitope = antigenic determinant.
Hint: Epi = “on top” → the part exposed to antibody

The specific site on an antigen that binds an antibody is called:

Antigenic determinant

What is another term for epitope?

Size and complexity
Larger, complex molecules (proteins) are more immunogenic.
Hint: Big + complex = immune system notices.

Which factor most strongly influences whether a substance is an immunogen?

Hapten

Classic example: drugs (e.g., penicillin).
Hint: Hapten = “helper needed.”

A small molecule that is not immunogenic alone but becomes immunogenic when attached to a carrier is:

Proteins or carbohydrates

In transfusion medicine, RBC antigens are typically:

Immunogen

It stimulated an immune response → immunogen.
Hint: If antibodies formed → it WAS immunogenic.

A patient develops an antibody only after transfusion. The antigen is best described as:

Antibody binds a specific site on antigen

Which scenario best represents an epitope-specific reaction?

It can bind multiple different antibodies

Multiple epitopes → multiple antibody binding sites.
Hint: More epitopes = more immune “targets.”

One antigen may have multiple epitopes. This means:

Hapten

Drug becomes immunogenic only when attached to RBC.
Hint: Classic “drug-induced hemolysis” setup.

A patient receives a drug that binds to RBC membranes and later develops antibodies. The drug alone is most likely a:

Epitope

A lab identifies an antibody reacting with only one part of an antigen. That part is the:

Polyclonal response

Multiple epitopes → multiple antibodies → polyclonal.
Hint: Mono = one epitope, poly = many.

A patient has antibodies to multiple epitopes on the same antigen. This is best described as:

Large protein with complex structure

Hint: Think: vaccines = proteins.

Which is MOST likely to be a strong immunogen?

They are less accessible or less foreign

Accessibility + foreignness matter.
Hint: If immune system “ignores” it → weak immunogen

Why are some RBC antigens weakly immunogenic?

Widely distributed and recognized as foreign

Which best explains why ABO antigens are highly immunogenic?

It cannot bind antibody effectively

No epitope = nothing to recognize

If an antigen loses its epitope structure, what happens?

Immunogenic antigen

Which mismatch is most likely to produce antibodies?

Immunogenic

A transfused patient develops antibodies 10 days later. The transfused RBC antigen must have been:

Epitope structure

A lab modifies an antigen so antibodies can no longer bind. What was most likely altered?

5 basic Ig molecules held together with a joinging (J) chain; pentamer

first responder; EFFICIENTLY activate complement

direct agglutination; immediate spin (IS) phase detection

IgM

bivalent; classic Y configuration

Key in HUMORAL immune response; infefficiently activates complement; CROSSES PLACENTA

inefficient in direct agglutination; ANTIGLOBULIN test detection

IgG

Primary

B cells are immune cells involved

smaller level of response

Ab produced in 5-10 days

IgM Immune response

MEMORY B cells are immune cells involved

LARGER level of response

Ab produced in 1-3 days

IgG Immune response

Strength of bond betw. Ab and Ag

Increases after each exposure

Higher in secondary (anamnestic) response

Due to affinity maturation

What is Ab affinity? In what stage of the immune response is Ab affinity higher? Why?

Classical is activated by Ag/Ab complexes

Ca and Mg

Anaphylatoxins, chemotactic, opsonization

How is the initiation of the classical complement pathway different from the alternative pathway? What ion must be present to activate the classical pathway? What are the biological effects of the activation of the complement cascade?

PRO = too much Ab

POST = too much Ag

bothat cause FALSE NEG

Prozone vs. Postzone

Temperature helps inc. the rate of the rxn.

IgG = warm temp = 37C = CINICALLY SIGNIFICANT

IgM = cold temp (room temp or below) = clinically insignificant

Why does temp matter in Ab/Ag rxns?

The lattice network of red cells may not form leading to a FASLE NEGATIVE interpretation

What would happen if the centrifuge speed is too low?

The shielding effect around the red cells are reduced allowing an inc. in Ab uptake

How does lowing the ionic strength of the test system affect agglutination rxns?

Finding units of blood when given prevalence

Relationship test - Direct vs. indirect exclusion

Immunogen

Foreign molecule that elicits an immune response

Blood group system

group of Ags in the RBC membrane that are related serologically and inherited though the same genetic inheritance pattern

dosage

variation in Ag expression because of the number of alleles present for a given trait

recessive

trait expressed in an individual only when inherited from both parents

Antigen (Ag)

foreign molecule that binds to an Ab or T-cell receptor

Law of independent Assortment

Genes on separate chromosomes behave randomly during meiosis

Phenotype

Determined by the presence or absence of hemagglutination of RBC Ags using specific antisera

Homozygous expression

Inheritance of 2 identical alleles for a given trait

Hardy-Weinburg equation

statistical formula to estimate the frequency of genetic diseases or observations of traits

Genotype

Genes inherited from each parent; determined through family studies or molecular testing

Heterozygous expression

inheritance of 2 different alleles for a given trait

Law of independent segregation

traits that are transmitted in a predictable fashion from one generation to the next

Epitope

antigenic determinant

dominant

trait expressed over another trait; requires inheritance of only one allele for expression

codominant

2 different inherited alleles are equally expressed in an individual

protiens are the best immunogens, then complex carbs

degree of foreignness - greater from self the more like it will cause an immune response

size > 10K Daltons

dosage and Ag density - # of RBCs and the amount of Ag they carry

route of admin - IM or IV injections are good at eliciting an immune response

What factors contribute to immunogenicity?

involves several WBCs

humoral - B cells receptors recognize foreign RBC Ags - B cell presents Ag to T cell - T cell cytokines tell B cell to turn into plasma cells that produce Abs (glycoproteins) with the same specificity as the B cell receptor - each plasma cell is a clone - Memory B cells also made at this time that can quickly respond if there is another exposure - Memory B cells dont need the T cell to be activated - think vaccination -

Describe the immune response in a transfusion setting

G - secondary immune response (anamnestic)

A - resides in mucus lining

M - primary immune response

D - Ag receptor on the naive B cell

E - activate mast cells = immediate hypersensitivity rxn

What are the 5 classes of Abs and their function?

pepsin and papain

What enzymes are used to divide Igs into the Fab and fc regions

heavy chain - variable and constant region that determines the class of Ag

light chain - variable and constant region made of only kappa and lambda

Fab region - Ag binding site

Fc region - determines the Ab function (contains complement binding region and cell activation region)

variable region - where the Ags bind on the heavy chain

hinge region - betw. Fc and Fab providing flexibility and 2 Ag binding sites

Ag binding site - Specificity determined by variable region that fits epitopes

macrophage binding site - opsonization and attaches to the Fc portion that signals activation = phagocytosis

Describe the regions of the antibody

extravascular hemolysis

Abs attached to RBCs signal clearance by liver and spleen

heavy chain - Mu / Gamma

light chain - kappa or lambda / kappa or lambda

J chain - yes / no

Molecular Wt - 900K / 150K

Valency - 10 / 2 (bivalent)

Total serum conc. - 5-10% / 70-80%

serum ½ life days - 5 / 23

crosses placenta - no / yes

activation of classical pathway of complement - very efficient (only needs 1) / not so efficient (needs 2)

Red cell clearance - intravascular / extravascular

detection in lab tests - IS = direct agglut. / AHG

structure - pentamer / monomer

exposure - acute or early / chronic or previous

IgM vs. IgG

intravascular hemolysis

complete activation of the classical pathway of complement and destruction of RBC

ABO is usually IgM

affinity maturation

the reason why Abs produce a stronger rxn after repeat exposure

IgG - Secondary Immune response

Anamnestic

w/in 1-3 days of exposure

higher conc. for longer because of memory B cells (larger # of plasma cells)

chronic or previous exposure

Lag after immunization - 5-10 days / 1-3 days

Peak response - smaller / larger

Ab isotype - usually IgM>IgG / Inc. IgG

Ab affinity - low avg. more variable / high avg. affinity maturation

Primary vs. secondary immune response

Immune complex

Antigen-Antibody Complex (combined Ab-Ag)

avidity

overall strength of rxn based on antigenic factor like size, shape and charge (goodness of fit)

overall stability and strength of the immune complex

influenced by Ab affinity and valency, size, shape, charge, noncovalent attractive forces

affinity

strength of binding betw. single Ab and epitope of an Ag affected by valency & fit of Ag

high affinity = binds quickly

Electrostatic forces (ionic bonds) - attraction based on opposite charges

hydrogen bonds - attraction of 2 neg charged groups for an H+ atom

hydrophobic bonds - weak bond from exclusion of water from the Ag-Ab complex

van der Waals forces - attraction betw. electron cloud (-) of one atom and the proton (+) w/in the nucleus of another atom

What are weak non-covalent bonds betw. Ab/Ag that hold the immune complex together?

transfusion rxn, anemia or HDFN (anemia and high levels of bilirubin in the fetus/newborn)

The destruction of RBCs result in what 3 thing?

alloantibodies

Abs produced in response to transfusion and pregnancies

Complement System

group of protein who’s responsible for antigen clearance, cell lysis and vasodilation

C1 - C9 circulate inactive as proenzymes - activation = active enzymes that enhance the immunological process

goal = opsonization, lysis of the target cells and stimulation of inflammatory mediators

C3→C3a (inflammation) & C3b (deposited on target cell for opsonization and phagocytosis)

C5→C5a (inflammation) & C5b →C9 = cell lysis

Complement proteins form membrane attack complex

Classical Pathway Described

Complement proteins react in specific seq. resulting in cell lysis

AB-Ag complex → C1 binds to Ab → activates C4, 2 then 3 → C1 splits C4 and C2 into a & b fragments → b binds to the cell and a enhances inflammatory response → C4bC2a complex forms called C3 convertase that converts C3 into active form which splits C3 into fragments → C3 convertase join with C3b to form C5 convertase which splits C5 → C5 to C9 = MAC causing lysis → MAC attaches to cell surface producing holes in the cell membrane and osmotic lysis

Alternative Pathway Described

Initiated by cell surface constituents

Results of Complement Activation

Anaphylatoxins - C3a, C4a and C5a complement proteins recruit phagocytes and promote inflammation, attach to mast cells and promote release of vasoactive amines (makes blood vessels permeable for fluid and cells to enter the area)

Chemotaxis - C5a attracts Neut. and Plt. to site of injury

Opsonization - C3b and C4b are Opsonin - molecule binds to Ag to promote phagocytosis & making the process most efficient → phagocyte + opsonin = really efficient phagocytic process → they also have a “c” & “d” fragment → d fragments can attach to red cells also

Mononuclear Phagocyte System

Present in secondary lymphoid organs - liver, spleen (largest), liver, lungs that clears old cells and ab-ag complexes

Stages of Agglutination

SENSITIZATION

Ab binds to Ag on the red cell membrane

recognition → antigenic determinants on RBC combine w/ Ag binding site on Ab → no visible agglut.

more Abs = better chance of collision events → relies on serum to cell ratio → add patient serum if rxn is weak

temp - IgG, 37C; IgM < or = 22C

Incubation - IS or after a specific time at 1-8C, RT or 37C

pH - 7.0

Ionic strength - can be adjusted with reagents

LATTICE FORMATION

Cell-Cell interactions - random collisions betw Ab coated cells develop cross-linkages = visible agglut.

Antigenic determinants bind with Ab binding sites on adj. cells (IgM because it is larger = direct agglutimation)

zeta potential - dist. bewt. cells caused by charged ions

zone of equivalence - Ag and Ab conc. → prozone = too much Ab = false neg → postzone = too much Ag = false neg

Centrifugation - time & speed to bring cells closer together → over = false pos & under = false neg

2-5% red cell suspension in salineis optimal

anti-I and anti-M

What antibodies are readily detectable when the pH is reduced?

Grading Agglutination

shake and tilt tube until button fully resuspended

0 - all red cells completely resuspended, red cells flow off button during grading

1+ - medium and small agglutinates - turbid background/many free cells

2+ - many medium agglutinates - clear background

3+ - several large agglutinates - clear background

4+ - RBC button is solid - clear background

HEMOLYSIS IS A POSITIVE RXN!

If activated by an immune complex → complement system → MAC → membrane damage → intracellular fluid released

Serum = active complement proteins

Plasma = anticoagulant binds Ca (needed to activate complement) → no complement activation

Abs to the LEWIS system Ags and Anti-Vel

What red cell Abs naturally display hemolysis?