Fundamentals of Immunology for Blood Bankers Flashcards
What is the immune system (IS)?
A controlled system of biological components (cells, tissues etc.) that work together to defend the body from invasion/infection.
What is immunity?
A specific response by the immune system to a previosly encountered pathogen via antigen-antibody complexes.
What are Cytokines?
- soluble protein and peptide molecules are powerful mediators of the immune system.
- comminucated between cells through plasma
How do Cytokines work? (3)
- bind to receptors on cells, # of receptors increase as cell is stimulated
- receptor and cytokine internalized, causes taret cell to differentiate
- cells respond to cytokines and react with chemoattraction, antiviral, antiproliferation, and immunomodulation
Name two cytokines?
- Lymphokines - produced by lymphocytes
- Monokines - produced by monocytes and macrophages.
What are Chemokines? (3)
- A type of cytokine
- A signalling protein produced that attracts leukocytes to the site of infection/inflammation. So immune cells can target and destroy invading bodies like microbes
- Form a concentration gradient; cells attracted to gradient and move toward higher concentration.
Compare natural immunity and adaptive (acquired) immunity?

See Pictures

What are the main components of Cellular and Humoral Immunity?
See picture
Humoral - Fluid Component: Complement (missing from table)

What are the Physical and Biochemical Barriers in natural immunity?

Acquired immunity: the antibody that the antigen is made against can be referred to as?
Antithetical Antigen
Antigen-antibody complexes are known to be _____ _______ _____ which do not allow the recognition of near misses.
Three dimensional interactions
What cells are responsible for humoral immunity?
B-cells
When b-cells are activated and start procuding antibodies, they are called?
Plasma cells

What do B cells do when they re-encounter a specific antigen?
What do they do when they encounter and unknown matching antigen?
- Multiple (clone itself)
- Internalizes antigen fragments, combines it with and presents it on its surface using its MHC II molecules.
Represent 60-80% of circulating lymphocytes and are the primary cells in cell mediated immunity.
T cells
What do T Helper (CD4) cells do? (4)
- Activate and direct the actions of other immune cells:
- Secrete cytokines that activate macrophages
- Activate B cells which make antibodies
- Recognise antigen with help of MCH II molecules

What do Cytotoxic T-Cells (CD8) do?
- Attack and lyse infected / damaged / dysfuctional cells

What do regulatory supressor T -cells do?
- Regulate the immune response
- inhibit activities of B and T cells to prevent immune system going out of control.
What are Natural Killer (NK) cells? (3)
- Granular lymphocytes that lyse virally infected cells, malignant cells and antibody-antigen complexs without needing stimulation.
- NK Cells act on cells missing self (MHC I)
- MHC I inhbits NK cells

What is the purpose of neutrophils?
- Phagocytosis
- Use granules in cytoplasm to break down ingested material and kill microrganisms
Where do basophils accumulate and what do they do?
What Ig are they activated by?
- Inflammatory sites
- release histamines
- IgE
What funstions do Eosinophils perform? (2)
- Kill invading microorganisms, incl. some parasites
- control allergic reactions by secreting histaminase, enzyme that breaks down histamine. Stops allregic reaction gettingout of control
Where are monocytes found?
- In the blood, when they enter tissues they become tissue macrophages
What are the two functions moncytes / macrophages perform?
- engulf and ingest antigents via phagocytosis
- Are Antigen Presenting Cells (APCs) that process and present antigens to lymphocytes during the immune repsponse.
What do APCs do?
- Display parts of ingested antigens bound to Major Histocompatibility Complex molecules (MHC) on their surfaces.
- certain T cells recognise this antigen-MHC complex and become active
What steps are involved in antigen presentation by APCs?
- antigen ingested and broken down
- vesicle w/ MHC molecules merges w/ vesicle w antigen bits, form antigen-MHC complex.
- Antigen-MHC complexes expoerted to surface
- CD4 / CD8 cells activated based on type of antigen-MHC complex exposed. MHC molecules help T cells recognise foreign antigens.
What part of the genome encodes MHC?
Human Leukocyte Antigen or Major Histocompatibility Factors
What is critical to MHC?
immune recognition and regulation of antigen presentation in cell-to-cell interations, transplantation, paternity testing, and specific HLA patterns.
Correlates with sucesptibiilty to certain diseases
What the classes of MHC molecules and their roles?
-
Class I -
- on all nucleated cells except trophoblasts (egg) and sperm, key in role in CD8 function.
-
Class II
- presemt on antigen presenting molecules like B-lymphocytes, activated T-cells, and dendritic cells.
What are the 5 classes of Igs?
- IgG
- IgM
- IgA
- IgE
- IgD
What is the function of IgG? (5)
- Can cross placenta - immunity to neonates
- neutralize toxins, fight infections
- Produced in response to antigens in transfusions / pregnancy
- Most involed in immune repose to antigens
- 80% of Igs are IgG
What is the function of IgM? (8)

- Antigenic receptor site on immature B-lymphocytes
- 10 bingding sites
- Neutralizes toxins
- Most effective antibody for agglutintion
- Activates complement w/ 5c region
- Largest, first and main antibody in primary immune response.
- 6% of Igs
What is the function of IgA? (4)
- Protect from bacteria and viruses
- Can cause anaphylactic transfusion reactions
- Secretory antibody
- 13% Igs
What is the function of IgE? (5)
- Moderates release of histamines and heparin from basophils and mast cells.
- Attaches to surface of basophils and triggers immune response
- responsible for immediate hypersensitivity/allergic reactions/parasite infections
- receptors blocked by antihistamines
- <1% Igs
What is the function of IgD?
- Dont really know
- may act as receptors for antigens when found on the surface of b-lymphocytes
What are antigens?
- Any substance capable of interacting with an antibody
- can cause an immune response
- contain antigenic determinants called epitopes.
What us an epitope?
The specific part of the antigen that is recognised by the immune system and part that binds w/ the antibody, b-cell or t-cell
Describe primary antibody structure?
- Y shape formed by:
- 2 large chains (inside)
- 2 light chains (outside arms)
- Bound by covalent bonds and disulfide bridges
What enzymes cleave antibodies into 3 fragments?
What are the 3 fragments antibodies can be cleaved into?
Where are the above sites located on the Y shape and what do they bind?
- Papain
- Pepsin
- 2 antigen binding fragments (FAB - Fragment Antigen Binding sites)
- 1 Crystalizable site (Fc - Fragment Crystalizable)
- FAB - top of antibody ( consists of heavy and light chain) - binds antigens
- Fc - bottom (heavy chains only), binds receptors on specific immune system cells
What is the variable domian of an antibody?
- Upper part of the FAB sites
- this area binds to s specific antigen
- 1 variable region at the top of each light and heavy chain.
What is the Constant domain and its role?
- This is the bottom of the Y on an antibody consisiting of both heavy chains.
- Activates complement cascade
What is the Hinge region?
- Center portion of the antibody
- lets antibody be flexible
- area where antibody can be broken apart by chemical treatment
What does the primary response depend on?
characteristics of the antigen and individual
IgM antibidy response proceeds in four phases after antigen challenge. What are the 4 phases?
- Lag - no antibody detectable
- Log - antibody titre increases logarthmically
- Plateau - antibody titre stabilizes
- Decline - antibody is catabolized
When does the secondary (amnestatic) response occur?
After repeated exposure to the same foreign substance.
- Stimulates proliferation of clonces of memory cells followed by corresponding antibody production.
How is the secondary response different from the primary response phases? (5)
- Shorter lag phase
- longer plateau
- more gradual decline
- IgG is main antibody
- Antibody levels are higher - 10X or greater
What is antibody affinity?
strength of attraction of antibody FAB site and expitope/determinant site on an antigen
What is antibody avidity?
The functional combining strength of antibody with its corresponding antigen.
strength of bonding is increased with multivalent antigen combined with more than one antibody combining activities.
To dissociate complex, all Ag-Ab bonds must be broken simultaneously
What are monoclonal antiodies?
Purified antibodies from a single cell.
- Very pure and very specific for a particular antigen
How were monoclonal antobodies produced?
- Fusing lymphocyes to produce a specific cell line that was immortal and produced specific antibodies. (Kohler, Mildstein, and Jerne 1975)
- Hybridoma (cell hybrid) (developed from myleoma (tumor) plasma cells)
- hybrid cells secrete antibodies characteristic of parent cell
- MAbs (monoclonal antibodies)
What is the greatest impact of MAbs in the analysis of cell membrane antigens in clinical applications? (5)
- Typing Tissue and Blood
- ID infectious agents
- ID tumor antigens and autoantibodies
- ID and quantifify hormones
- delivering immunotherapy
What is complement?

A complex group of > 20 circulating and cell membrane protiens, protienases, and other enzymes.
The larger fragment is designated “b” and the smaller fragments as “a” EXCEPT for C2 which is the other way around.
Most of the proteins are normally inactive, but in response to the recognition of molecular components of microorganisms they become sequentially activated in an enzyme cascade – the activation of one protein enzymatically cleaves and activates the next protein in the cascade. Complement can be activated via three different pathways
What are the primary roles of complement? (5)
- Direct lysis of cells and bacteria
- Oposonization
- phagocytosis
- Makes fragment split products involved in: inflammatory responses
- Memebrane Attack Complex (MAC)
What are the three pathways that acivate complement protiens through a cascade event?
- Classical
- Alternative
- Lectin
Where do the 3 complement cascade pathways converge?
What proteins are common to all 3 pathways?

- Pathways converge at the point of cleavage of C3 to C3b
- C2 and C5 are common to all 3 pathways

What are three techniques to detect antibodies or antigens?
- Hemagglutination (precipitation)
- Agglutination inhibition
- Hemolysis
What are the antibody or antigen tests that use radioisitope, enzyme or flourescent labels? (4)
- Radioimmunoassya (RIA)
- ELISA
- Western Blot (WB)
- Immunoflourescence (IF)
Hemagglutination Technique: What are Red cell agglutination reactions used for?
- Analalyse blood group antigen-antibody responses
- typing ABO, Rh,a nd other blood group antigens
What are the two stages of Red cell agglutination reactions?
-
Sensitization
- Antigen-Antibody binding
- Epitopes on RBC membrane bind w/ FAB region
- No Agglutination visible
-
Precipitation
- Lattice structure composed of antigen-antibody bridges
- Visible agglutination present
Describe the Agglutination Inhibition Method?
What is this test useful for?
- Substrate added to reagtion that blocks agglutinate formation (antibody-antigen complex)
- Secretory studys to check for ABO subtances prescence in body fluids and secretions
Describe the Hemolysis method?
Give an example?
- String positive result indicates formation of antibody-antigen complex fixed by a complement system resulting in RBC lysis
- Lewis blood group antibodies:
- Anti-Lea
- Anti-Leb
What intiates and immune response?
- Presentation of antigen (initiates formation, reacts with antibody)
- Immunogen (initiates immune response)
What is primarily detected in blood banking?
- Detection of antibodies to blood group antigens
What factors influence antigen-antibody reactions? (4)
- Intermolecular binding forces
- antibody properties
- Host factors
- tolerance
What cell assists in antibody-dependent cell mediated cyctotoxicity (ADCC)?
lymphocytes
What are host factors?
Factors unique to a host that determine an individuals immune response:
- Age
- Sex
- Hormones
- Nutritional status
- Race
- Exercise
- Disease/injury
What factors influence the first stage of agglutination and must be controlled by a technician? (6)
- Centrifugation
- Temperature
- pH
- Incubation time
- type of enhancement media
- Zonal reactions
Temperature:
- IgM react best at what temperature range?
- IgG are better detected at what temperature?
- React best 4 - 27 oC (immediate spin phase)
- ABH, I, M, N, Lea, Leb, P1
- 37 oC with AHG phase
- D,C, E, c, e, K, Fy, Jk, S, s, Lea, Leb
- Antibodies that react in vitro below 37 oC = no clinical significance because rarely destroy transfused cells
Why is centrifugation of RBCs and important factor?
- Having RBCs iin closer togther they overcome zeta potential (repel eachother)
- increased antibody-antigen formation
- Better agglutination
What are 3 kinds of enhancement media and their effect on test?
- LISS - low ionic strength, RBCs take up antibody quickly 15 - 30 mins at 37oC
- PEG (polyethylene glycol) - increases test sensitivity, 10 - 30 mins at 37oC
- 22% Albumin - causes agglutination by adjusting zeta potential between RBCs 15 - 60 mins at 37oC
What is the ideal pH for agglutination reactions?
ideally 6.5 - 7.5
What are the Zonal Reactions?
Various antibody to antigen ratios:
What is the Zone of Equivalence?
Optimum antigen and antibody concentration present
What is the Prozone Reaction?
False negative reaction due to excess antiobody
What is a postzone reaction?
False negative due to excess antigen
What Ig types are important in blood banking?
- IgM
- ABH, MN, Lewis (Lea and Leb), Lutheran (Lua), and P blood group systems
- IgG
- Ss, Kell (Kk, Jsa, Jsb, Kpa, Kpb, Lutheran (Lub), Duffy (Fya, Fyb) and Kidd (Jka, Jkb) antigens
What is Hypersensitivity?
An inflammatory response to foreign antigens; can be cell or antibody mediated.
What is Type I hypersensitivity? (4)
- Anaphylaxis or immediate hypersensitivity
- histamine released by mast cells / basophiles w/ IgE
- IgE made against antibodies
- Occurs in IgA deficient individuals who recieve plasma w/ IgA
What is Type II Hyper sensitivity? (3)
- Involves IgM or IgG w/ complement, phagocytes, and proteolytic enzymes.
- Examples are:
- HDN
- Transfusion reactions
- Caused by blood group antibodies and autoimmune hemolytic reactions.
What is Type III hypersensitivity? (2)
- Involves IgG + Phagocytes and IgM + complement
- Results in tissue damage from:
- Immunue complexes of antigen-Ab aggregates
- Complement
- Phagocytes
- Penecillin and oth. drug induced antibodies can lead to HTRs
What is Type IV hypersensitivity? (3)
- T-cell mediated and their cytokines
- fatal if untreated
- GRAFT VS. HOST (most common cause)
- Immunocompromised and immunosuppresed patients must received iraadiated blood products so t-lymphocytes do not engraft and attack host tissues
- bone marrow transplant recipients
- stem cell recipients
What are monoclonal and polyclonal gammopathies?
- Plasma cell neoplams cause abnormal Igs from single b-cell clone (mono)
- Plasma cell neoplams cause abnormal Igs from multiple b-cell clones (poly)
****multiple myloma patients demonstarte rouleux during testing
Hemolytic Diseases of the Newborn (HDN): Severe HDFN is most often associated with which antibodies?
IgG
What are the primary phagocytic cells?
- Polymorphonuclear neutrophilic (PMN) leukocytes
- Mononuclear monocytes-macrophages
What is the process of phagocytosis? (4)
- Chemotaxis
- Adherence (opsonization)
- Engulfment
- Digestion
- Subsequent phagocytic activity
What are the only imunologically specific cellular components of the immune system and the adaptive components of innate immunity?
B Lymphocytes and T Lymphocytes
What makes up primary (central) lymphoid tissue?
- Bone marrow
- (and/or fetal liver)
- Thymus
What are the secondary lymphoid tissues? (8)
- Lymph nodes
- spleen
- GALT (guts Associated Tissues)
- BALT (bronchus associated tissues)
- SALT (Skin assoicated tissues)
- Thoracic duct
- Blood
- Mature lymps and accessory cells ( i.e. APCs)
Most circulating lymphocytes are ____ , they are responsible for cellular immune responses and involved in the regulation of antibody reactions in conjunction with ____.
- T lymphocytes
- B lymphocytes
What are the subset of B cells?
How are they distiguished?
- B1 and B2 cells
- B1 cells are distiguished by the CD5 marker, self renewing set, respond to common microbe antigens, and sometimes generate autoantibodies
B cells are derived from an antigen-independent maturation process occuring in?
Bone Marrow
GALT
These cells are not normally found in circulating blood, but in the bone marrow in cencentrations no higher than 2% normally. What are they?
Plasma cells
B and T cells mature into _____ _____, the functional unti of the immune system.
Effector Cells
What are the four unique serum proteins found in the alternative pathway?
- Factor D
- Factor B
- Factor P (Properdin)
- IF (initiating factor)
What are the inhibitory proteins that regulate complement? (8)
- C1 Inhibitor (C1NH)
- Factor H
- Factor I
- C4-Binding Protien (C4BP)
- Anaphylatoxin inhbitor
- Anaphylatoxin inactivator
- MAC inhibitor
- C3 Nephritic Factor (NF)
What are the physiological consequences of complement activation?
- Blood vessel dialation
- Increased vascular permeability
- Cellular consequences:
- **Hemolysis** most impt in blood banking, concern is cell membrane lysis of antibody coated targets
- Opsonization facilitating phagocytosis
- Mediating hypersensitivity
- Cell activation productig inflammatory mediators
- Cytolysis
Complement can be elevated in many inflammatory conditions and is therfore not clincally significant.
Low levels of complement suggest what biological effects? (3)
- Complement excessively activated recently
- Complement is currently being consumed
- Single complement component missung due to genetic defect.
Complement is analysed by a _____ _____ ______ called __________?
Evaluation of which components is most useful in hemolytic transfusion reactions and autoimmune hemolytic anemias?
- Serum/plasma assay
- Nephelometry
- C3b and C3d
What are non-specific indicators of an inflammatory response?
Acute-phase reactants
Acute phase reactants include? (4)
- C-reactive protiens (CRP)
- Inflammatory mediators (C3 and C4)
- Fibrinogen
- Transport protiens (haptoglobin)
- Inhibitors (alpha1-antitrypsin)
- a1-acid-glycoprotein.
Why is C-reactive protein an acute phase protein of choice for testing?
- Changes show great sensitivity
- Changes independent of ESR and parallel to inflammation
- direct and quantitative measure of acutephase reation, due to its fast kinetics, gives info on actual clinical situation.
CRP is valuble monitoring therapy and diseaseactivity in?
- Rheumatoid arthritis
- Bacterial infection (septicemia)
- Rheumatic fever
- Crohn’s disease
- myocardial infraction
- stronger predictor of cardiovascular events than LDL Cholesterol
- Elevated can signal infection much earlier than bacterial culture confirmation
The C reactive protein agglutination test is based on?
Reaction between patient serum containing CRP as the antigen and corresponding antihuman CRP antibodies coated to the treated surface of latex particles
What lead to the development of highly specific and sensitive assays?
Monoclonal antibody (MAbs)
The specimen of choice for most immunology tests is?
Serum, but oth. body fluids may also be tested
Blood specimens are collected when and why?
Before a meal to aviod chyle (emulsion of fat globules) and appear in serum after eating / digestion.
What contamination must be avoided because these substances have a denaturig effect of seim proteins?
Acid or alkali
What urine specimen is preferred for pregnancy and UTI tests?
Mid-stream clean catch
Urine for hCG tests should be collected at a suitable time interval beacuse?
What happens to concentration of hCG after conception?
When are peak levels reached?
hCG levels need to rise to a detectable level.
- hCG doubles every 2 - 3 days with [serum] level reaching 10 - 50 mIU/mL in the week after conception
- Peak levels reached 2-3 months after last period
Why is complement inactivation done? (3)
- complement is known to interfere with reaction of certain syphilis tests and complement components (i.e C1q).
- It can agglutinate latex particles = false positive
- can cause lysis of indicator cells in hemagglutination assays.
How is complement inactivated?
- Heat at 56oC for 30 minutes
- When >4 hrs since inactivation done; reinactivate speciment by heating to 56oC for 10 minutes
What is the principle of agglutination tests?
Precipitation and agglutination are the visible expression of the aggregation of antigens and antibodies via the formation of a framework of alternating antibody and antigen molecules.
Precipitation is the term for?
Aggregation of soluble test antigens
What is agglutination?
Describles the aggregation of particulate test antigens
What are some examples of artifical carriers?
- Latex particles and colliodal charcoal
- Cells unrelated to antigen, such as erthrocytes coated with constant amount of antigen can be used as biological carriers.
- Whole bacterial cells with antigen that will bind with antibodies in repsonse to that antigen when intorduced to the host.
What factors does the quality of agglutination tests depend on? (3)
- TIme of incubation of antibody with source (serum)
- Amount of avidity of antigen conjugated to carrier
- Conditions of test environment (pH, [protein])
Describe latex agglutination? (3)
- Abs bount to surface of latex beads (### exposed antigen binding sites)
- If antigen present (i.e. CRP), it will bind to latrex beads forming visible cross-linked aggregates.
- In some systems (pregnancy, rubella, Abs testing) latex particles coated w/ antigen; in prescence of serum Abs these particles agglutinate into large visble clumps.
What are the two subunits of hCG and which one is tested for and why?
- Alpha and Beta
- Beta tested for because the alpha subunit is also found in FSH, LH, TSH as well.
- Beta subunit has a unqiue carboxy-terminal region, so tests have MAb against beta unit to increase specificity and cutt down cross reactivity.
What are flocculation tests based on?
Why are is this test macroscopically and microscopically visible?
- Interaction between souble antigen w/ antibody particles, redulting in formation of fine particles.
- macroscopically and microscopically visible because precipitate is forced to remain in a confined space.
Floculation testing is used in testing for what?
Name the tests and the antigen antibody process leading to flocculation? (2)
- Syphilis testing
- VDRL
- antibody like protien regain binds to test antigen - cardiolipid-lecithin-cated cholesterol particles ==> particles flocculate
- Rapid Plasma Regain (RPR)
- cardiolipid-lecithin-cated cholesterol particles with choline chloride contains charcoal = macroscopically visible flocculation.
- VDRL
What is Direct Bacterial Agglutination used for?
- Used to detect antibodies against the pathogen
- measure antibody produced by host to the antigen on the surfact=e of bacteria in reponse to infection with it.
- bacterial clump together in visble aggregates
What influences the formation of aggregates in solution?
Electrostatic and other forces
Why is sterile physiologic saline used for bacterial agglutination?
sterile physiologic saline with free positive ions used enhances aggregation of bacteria because most bacteral serfaces have a -ve charge that causes them to repel eachother.
Bacterial Agglutination: Why is tube testing better?
- More sensitive than slide testing
- allows more time for antigen-antibody reaction
- small volume of liquid in slide testing can evaporate quickly
What does hemaggutination test?
Antibodies to erythrocyte (RBC) antigens
same principle as latex agglutination
What does centrifugation do?
Physically forces cells togther
Agglutination enhancement: What does treatment with proteolytic enzymes do?
Alters the zeta potential or dielectric constant to enhance the chances of demonstratable agglutination.
Agglutination enhancement: what does the use of Colliods do?
Alters the zeta potential of cells so IgG will be able to agglutinate
Agglutination enhancement: When might Antihuman Globulin (AHG) Testing be used?
To facilitate agglutination if other tech. do not work especially if antigens deeply embedded in cell membrane surface.
Graded Agglutination reactions?

What is the priciple of the hCG test?
Agglutination between latex particles coated with anti-hCG antibodies and hCG
What is a positive hCG test?
What is a negative?
What can caise false positives?
- Aggltination w/i 2 mins
- No agglutination seen
- hCG injection
- chrioepithelioma
- hydratiform mole
- Excessive asprin ingestion