chapter 1 Flashcards
antigens
substances that can be recognized as foreign by the body, may act as immunogens capable of triggering an immune response( can be more or less immunogenic); will be on RBC’s or other cells
antibodies
also known as immunoglobulins(Ig); proteins that attack foreign antigens by attaching to a specific epitope or antigenic determinant.
epitope
specific section on an antigen that an antibody binds to, an antigen can have multiple different types of epitopes.
immunogenicity
how readily an antigen elicits the formation of an antibody
factors affecting immunogenicity
in general:
chemical composition
- the more complex amino acid= more immunogenic
- proteins are more immunogenic than carbohydrates ( exception is A& B antigens are carbohydrates )
size
-larger antigens are more immunogenic
degradability
-antigens that quickly degree are unstable snd less immunogenic
foreignness
-the more dissimilar the antigen id the more immunogenic
in general an antigen is more aimmunogenic if it is …
a protein
big
stable
different
in general antigens are less immunogenic if they are…
carbohydrates
small
fragile (degrades)
similar
there are exceptions ex) ABO are carbohydrates
what cell is of most concern for blood bank
RBC’s surface antigens are of most concern because they have the greatest clinical significance. more immunogenic
what do we transfuse
RBC’s
plasma
platelets
WBC’S
What do we not intentionally transfuse
WBC’S, they are filtered out of blood of blood but a few may remain.
Explain an immune-mediated hemolytic transfusion
if a patient is transfused with RBC’s that express an antigen for which the patient already developed an antibody with the corresponding specificity they may cause a significant immune response.
passive immunity
when a person is given antibodies rather than producing them through their immune system. May occur Naturally ( ex. cross placental transfer of antibodies from mom to baby) or by Artificial infusion of plasma or plasma derived immunoglobulins.
active immunity
results from exposure to a disease triggering the immune system to produce antibodies to that disease.Can be acquired naturally through exposure and infection by a pathogen or artificially through a vaccine.
key role in human immune system
to distinguish self from non self and eliminate non self. it also eliminates cancerous tutor cells as well as senescent (old) cells.
innate ( natural) immune system
acts within minutes of exposure
present at birth
nonadaptive
doesn’t have immunological memory ; magnitude of response doesn’t ever increase
defence : anatomical barriers such as skin, mucous membranes & respiratory tract cilia. and presence of indigenous microbiota stops pathogens from growing in their place.
cell mediated immunity
immune response from leukocytes that does NOT involve the production of antibodies
PAMPs
pathogen associated molecular pattern
innate cell-mediated immunity recognizes PAMPs by pattern recognition receptors present on dendritic cells, macrophages, monocytes and neutrophils.
these phagocytes them engulf the identified pathogen
natural killer (NK) cells
NK have a key role in innate immunity; they are able to identify and destroy cells that lack major histocompatibility complex (MCH) class 1 expression. when a cell lacks MCH 1 it is an abnormal cell that may be virally infected or cancerous which is suppressing the MCH 1 expression in this case NK lymphocytes will be stimulated to release cytolytic granules, inducing apoptosis in the affected cells
MCH 1 and MCH 2 are found where
MCH 1 are found on the surface of all normal nucleated cells of the body. The complex displays peptide fragments from proteins found within the cell, which will be presented to cytolytic (CD8+) T cells.
MCH 2 are found on on antigen presenting cells ( B cells, dendritic cells and other phagocytes). They are for presenting antigen obtained from extracellular pathogens to helper (CD4+) T cells.
acquired immunity
revolves around B and T lymps and their and their interactions with antigen presenting cells. If a cell is infected intracellularly with a pathogen the cell will incorporate peptides from the pathogen into their MHC 1 molecule and cytolytic (CD8+) T cells with the receptor specific to a foreign antigen can destroy the cell.
cytokines
proteins that include interferons, interleukins, tutor necrosis factor, transforming growth factor beta and other chemokines. various cytokines may function to attract leukocyte migration( chemotaxis) & incite inflammation, causing vasodilation and localized heat, pain, renders & swelling.
all of this assist phagocytes in destroying pathogens
cytokines also bridge the gap between cellular and humeral immunity, because some stimulate antibody production
Humoral immunity
immunity that is mediated by macromolecules; especially the production of antibodies, Complement activation, & production of host defence peptides
- complement is an innate humeral immunity
antibodies are an adaptive humoral immunity
primary antibody response
immunoglobulins act as B cell receptors and Igs with a singular antigen specificity are attached to the surface of mature naive B lymphs as they enter the blood stream from the bone marrow if their receptor binds the corresponding antigen they become activated
Burnets clonal selection theory
mechanism of clonal deletion , self -reactive lymphs are eliminated, ensuring only B cells with receptors to foreign bodies can activate
B cells become memory cells and plasma cells
they display the pathogen material in MHC 2 (strictly for presenting antigens from extracellular pathogens to helper (CD4+) T cells)
Helper T cell stimulates B cel to divide into identical clones and produce some Memory cells and mostly plasma cells.
memory cells
help accelerate immune resumes upon subsequent exposures to the same antigen
plasma cells
capable of secreting antibodies into circulating blood. each plasma cell clone secretes only antibodies of the same antigen specificity as the initial activated B cell
IgM & IgG after exposure ( class switching )
initially IgM are more present on B cells for the first few days then it decrease while IgG increases and is the most; over time they gradually decrease but once a second exposure occurs IgG immediately increases rapidly due to memory cells stimulating a faster and stronger response
somatic hypermutation
during activation some B cells immunoglobulin mutations result in increase affinity for the antigen
3 main roles of antigen -antibody couples
- neutralization
- activation of complement (intravascular hemolysis)
- opsonization ( extravascular hemolysis )
structure of immunoglobulins and enzyme breakage
two identicslheavy chains are connected together each with a light chain on each side (identical)
top of the antibody is the Fab portion and is variable, it contains the two binding sites
bottom is the Fc portion and is constant
disulfide bonds hold all the chains together
if treated by enzyme papain the antibody will be cleaved at the hinge region above the disulphide bonds connecting two heavy chains
-this creates two fab sections and a Fc section all separated
antibody chains determined by class
the two light chains of all antibodies can be either kappa or lambda
the heavy chains depend on which of the 5 classes the antibody belongs to I gA ( alpha) IgD( delta) IgE ( elipson) IgG ( gamma ) and IgM ( mu)
IgM
mainly found in blood and lymph
bound to the surface of naive B lymphs along with IgD
have 5 monomers linked by J chains, making 10 binding sites ( really good at complement )
can be crab shaped
large structure makes it good at direct agglutination of red cells
reacts best at colder temps ( room temp or colder )
after immediate spin
however some have a broad thermal range of activity and can react at warmer temps
exothermic reaction ( releasing free heat energy)
antibodies against carbohydrate antigens tend to be IgM ( ABO, M,N,lewis etc)
compromises approx. 10% of Igs in plasma (secondment abundant )
IgG
principle isotope in blood and extracellular fluid (75%)
reacts best at body temperature ( 37 degrees)
Antibodies against protein antigens tend to be of class IgG
undergo endothermic reactions ( absorbing free heat energy)
small monomers with 2 antigen binding sites
cannot usually cause direct agglutination
there are 4 subclasses of IgG each slightly caring in structure ( IgG1, IgG2, IgG3, IgG4)
IgG1 & and IgG3
strongly activate complement and are readily recognized by bound macrophages .Both are capable of crossing the placenta from mother to baby
therefore IgG1 and IgG3 are the most clinically significant subclasses of IgG
IgG2
weakly activates complement, is less readily recognized by the placenta and cant effectively cross the placenta
IgG4
does not activate complement
show little to no macrophage recognition
but can cross the placenta
IgA
principle isotype in mucosal secretions
produced by specific plasma cells residing just below the basement membrane of secretory epithelium in the lamina propia
IgA is secreted as a dimer: two monomers linked by a J chain
IgA doesn’t activate complement
IgE
a monomer that can activate mast cells (in tissue) and basophils ( bloodstream) causing allergic reactions
IgD
IgD is a monomer found predominantly bound to the surface of naive B lymphocytes along with surface IgM function not understood
DTT and 2-ME
dithiotgreitol (DTT) or 2-mercaptoethanol (2-ME) can selectively cleave the disulfidebonds of IgM but leave IgG intact
IgMs found naturally
IgM against RBC antigens tend to to be found in patients without any exposure to red cells with corresponding antigen.
This is due to environmental exposure to similar or identical carbohydrates in nature
such as thought food or contact with microorganisms
IgG usually require RBC with corresponding protein before alloimmunization can occur
this usually happens through transfusion or pregnancy
law of mass action
a large number for Ko corresponds with a strong avidity , meaning antibodies bind quickly and doesn’t break apart easily
once an antibody & antigen bind, they can dissociate and bind over and over until equilibrium is reached
Ko= ka/kd
Ko is proportional to concentration of antigen- antibody complex
affinity vs avidity
affinity is the bond between one antigen and antibody. this reflects the initial attraction towards the epitope
avidity is the sum of all attractive forces between antigens and antibodies which keeps antigens and antibodies together once binding has occurred
in vivo avidity is more relevant because most antigens are multivalent, increased avidity can compensate for decreased affinity
two stages of agglutination
sensitization: occurs when antigen and antibody bind to form an immune complex
in the second step, aggregates develop through lattice formation which allows agglutination to be seen
lattice formation: cross links form between red blood cells creating lattice that allows visualization of antigen -antibody reactions
things that affect sensitization
temperature :IgM prefer cold temps, IgG perfer warm temps
generally clinically significant antibodies react at 37 degrees however this is not always true because ABO( the most clinically significant) reacts at colder temps ( 4degrees - rm temp )
incubation time: sufficient time is needed for antigen-antibody sections to reach equilibrium
the saline test without enhancements generally takes 30-60 mins of incubation at 37 degrees
generally those involving antisera containing IgM
may not require incubation ( read at immediate spin)
pH: pH of 7 is typically used, some anti- M in patients plasma has preferred a lower pH but antibody detection errors may occur if acidified aline is used routinely
ionic strength: isotonic saline ( 0.85%) dissociates into Na+ and Cl-
RBCs carry a net neg charge so Na+ are attracted to it. This makes RBCs harder to get close to one another bc the same charges will repel each other this is called the zeta potential
Low ionic strength saline (LISS)
when ionic strength is decreased the rate of interaction between antibodies and antigens is increased
this can be achieved using low ion strength saline (LISS)
However - antibodies especially complement can non specifically bind to RBCs id the ionic strength is too low, causing false positive reaction
plasma to cell ration
& addition of plasma/red cells
prozone: l antibodies in excess
- too many antibodies and not enough antigens, cant bind properly, incapable of sensitization
equilibrium: agglutination occurs
~ 1 drop of RBCs (2-5%)
~ 1-3 drops of plasma
postzone: antigen excess
while the detection of weakly reacting patient antibodies may ne enhance by additional drops of plasma; additional drops shouldn’t be added when using LISS
altering serum to cell ratio beyond manufacturers instructions will modify the ionic strength and decrease sensitivity
the addition of red cells is not likely to enhance reaction
Polyethylene glycol (PEG) with weak reactions
PEG can be added to increase sensitization
it will remove water increasing the concentration of antibodies and increasing uptake
anti-human globulin ( AHG)
AHG is added to sensitized RBCs that have been washed multiple times, they bridge the gap between RBCs by attaching tp the Fc portion of IgG and forming a lattice
Chemically modified anti-D
treated with sulfhydryl agents to split disulfied bonds between heavy chains allows the Fab fragment to span a wider distance and cross link antigens
enhancing second stage of agglutination
proteolytic enzymes: decrease the neg surface charge of RBCs however this process can also destroy certain antigens
positively charge polymers: cause spontaneous agglutination in RBCs, bringing them close enough for IgGs to cross link then sodium citrate- glucose is added to disperse the cells but crosslinks will remain
bovine albumin: modifies dielectric constant to disperse zeta potential and increasing viscosity which brings RBCs closer together
22% bovine albumin can be added as an enhancement medium
6% bovine albumin can be used as a control medium
Centrifugation: can sometimes overcome zeta potential enough to push RBCs closer together
Membrane Attack Complex (MAC)
Complement is part of the innate immune system
it causes cell destruction either indirectly through C3b attachment or directly through the formation of membrane attack complex
usually complement stops at C3b but if enough is produced quickly before inactivation it will begin to form a MAC
first C3b binds to C4b2a = C4b2a3b aka C5 convertase
C5 convertase enzyme cleaves C5 into C5a( a potent anaphylatoxin) & C5b ( binds to cell)
C6 &C7 bind to C5b inserting into the lipid bilayer
C8 binds and creates a small pore in membrane
after the addition of multiple C9 monomers, a tubular structure is formed that allows water and solutes to pass freely through the membrane; sodium and water enter cell and cause lysis and swelling
regulated by complement regulation proteins to prevent rampant cell destruction
- C5b67 can be inactivated by vitronectin
- if C8 binds to a fluid phase C5b67 it cannot insert into the cell membrane
- CD59 (protectin) and a homologus restrictive factor can bind to C8 and block insertion of C9 into cell membranes
3 Complement pathwayS
The goal of all pathways is C3b
classical pathway: presence of antibody -antigen complex activates complement
linked to adaptive immune system
alternate pathway : a natural defence system; specific antibody presence is not required . Linked to innate system
Lectin or mannose binding pathway :
classical pathway
complement is activated ( usually by IgM)
the recognition unit (C1) made up of C1q and 2 of each C1r and C1s, C1q is shaped like 6 tulips
-this binds with a constant region of Ig complexed with an antigen
- C1 is Ca+ dependent
C1 cleaves C4 into C4a & C4b
- C4a floats away to cause inflammation (anaphylactoxin)
- C4b attaches to cell and acts as binding site for C2 (C2 binding is Mg2+ dependant )
C1 cleaves C2 into C2a & C2b
- C2b floats away to cause inflammation
- C2a remains bound
making C4b2a= C3 convertase
C3 convertase cleaves C3 into C3a & C3b
- C3a floats away to cause inflammation
-C3b is capable of binding if close enough to C4b2a
this will further complement activation
* one C3 convertase can convert 200 C3 molecules
Classical pathway regulation
keeps complement activated by classical pathway in check
fluid phase: C1 inhibitor inactivates C1r & C1s
C4 binding protein ( C4bp) catabolizes C4b and promotes dissociation of C2a from C4b2a
**factor I inactivates C3b by cleaving bound C3b into C3c ( floats away) & C3d which remains permanently bound to the cell FOREVER.
cell surface : complement control proteins decay acceleration factor (DAF) & complement receptor 1 (CR1)
these work to:
-inhibit binding of C2 to C4b
- disassociate C2a from C4b known as “decay acceleration”
-promotes catabolism of C4b by factor 1
lectin pathway
homologous to the classical pathway but activated without an antibody .
Mannose-binding lectin(MBL) a molecule belonging to the same family as C1q, binds to bacteria and interacts similar in structure to C1r & C1s to activate complement
alternative pathway
also called amplification loop
usually triggered by something other than an immunoglobulin
innately responds to charged and neutral sugar targets
a small amount of C3b is continuously produced from free floating C3 through a process called spontaneous ticker activation
if C3b binds to unaltered self structure, further activation is inhibited by factors I &H
If C3b binds to a surface such as a pathogen or altered host cell more C3b will be amplified by factors B& D
