Exam 1 Study Guide
1. Know specificity, avidity, affinity
a. Specificity: 1 antigen is specific for 1 antibody
b. Avidity: overall strength of reaction between several epitopes and antibodies i. Dependent on:
1. Affinity of antibody 2. Valency
3. Non-covalent attractive forces
4. Expresses the binding strength of a multivalent antigen with antibodies
5. Measures functional affinity of antiserum for the whole antigen c. Affinity: strength of binding between single antibody and epitope of an
antigen. Strength of a single antigen-antibody bond produced by the summation of attractive and repulsive forces.
2. Know hydridoma technology and monoclonal reagents a. Polyclonal
i. Antibodies produced by B cells are specific to one epitope of an antigen. In order to be most effective the immune system produces multiple antibodies to the same antigen using multiple B cell clones. ii. Manufactured by stimulating a donor to produce immune antibodies iii. Different epitopes on a single antigen induce the proliferation of a
variety of B-cell clones, resulting in a heterogeneous population of serum antibodies.
iv. Produced in response to a single antigen with multiple epitopes v. Improves the immune response in respect to quality and quantity.
Antibodies against more than one epitope is required to provide immunity against an entire antigen
vi. But can vary in antibody concentration b. Monoclonal
i. Are directed against one specific epitope of an antigen. ii. Gives consistency and reliability
iii. Produced using hydridoma technology – isolate individual B cells and propagate in cell culture
iv. Highly specific, well characterized, uniformly reactive c. Hydridoma Technology/Mouse spleen
i. First the mouse is injected with what ever you want to make an antibody against.
ii. Then the mouse is killed and his spleen is removed, ground up. iii. The spleen is rich in B cells or normal antibody forming cells or
lymphocytes.
iv. The “oma” cells are purchased and added with the B cells in the presence of PEG and placed onto HAT media
v. What happens? The tumor/oma cells that fuse together will die. WHY? The spleen cells that fuse with each other die eventually. Why? BUT the hybridoma of Tumor and Bcells live. WHY???? vi. The hybrids grow and grow.
vii. The clones are tested and one is selected to grow more and produce the antibody.
3. Where do you find antigens?
4. What makes a good antigen?
a. Many others but these are the main 5- also charge, solubility, Accessibility, digestability
i. Foreignness- Must be identified as non-self
ii. Size- larger than 10000 daltons (smaller would be considered hapten) iii. Dosage- amount of substance (rbc) and number of ag sites on each
cell
iv. Route- Injection in to blood stream is most likely to cause response v. Composition- Protein Carbo Lipids
1. More complex glycoprotein and glycolipid is more immunogenic a. Proteins – Rh, M, N
b. Glycolipids – ABH, Lewis, Ii, P 5. Define Antigen and Antibody
a. Antigen: Non-self
b. Antibody: Immune response to non-self
6. Know antibody classes important to blood banking – IgG vs IgM a. IgM
i. contains 10 potential ag-binding sites ii. 5 basic Ig units held together by J chain
iii. directly agglutinate red cells in saline due to large size iv. ABO antibodies are IgM and can cause rapid hemolysis
v. Lewis, Ii, P, MNS common IgM abs vi. RT reactivity
vii. Can be destroyed by sulfhydryl reagents – destroys disulfide bonds viii. 5-10% Serum Ig
ix. Activates classical pathway b. IgG
i. Not likely to cause rapid hemolysis from complement activation
ii. Consists of 2 gamma heavy chains and 2 light chains – either kappa or lambda
iii. Bivalent – has 2 ag-binding sites
iv. Not efficient in agglutinating red cells in saline
v. Fc receptors on placental cells allows passage across the placenta – protects
vi. Against infection but can also lead to HDN
vii. Requires 2 IgG molecules to activate complement viii. 4 subclasses: IgG1, IgG2, IgG3, IgG4
ix. Rh: usually IgG1 and IgG3 x. Kell and Duffy: usually IgG1 xi. Kidd: usually IgG3
xii. Most clinically significant, reacts at 37 degrees xiii. Can cause Trans Rxns and HDN
7. Know immune system categories: Innate vs Acquired a. Innate
i. Natural – present at birth ii. Primary line of defense
iii. Early evolutionary development iv. Nonspecific
v. Immediately available
vii. NO MEMORY – same exact response time after time viii. Others
1. Physical: Skin, mucus membrane, normal flora, cilia, cough reflex
2. Chemical: Secretions, low pH of stomach 3. Cellular: Phagocytic cells
4. Humoral: Complement alternate pathway, cytokines 5. Inflammation
b. Acquired
i. Adaptive, secondary ii. Supplements innate
iii. When invading foreign substance or pathogen evades innate response iv. Later evolutionary development – only seen in invertebrates
v. Specific
vi. Acquired by contact with specific foreign substance
vii. Initial contact with foreign substance triggers synthesis of specialized viii. Antibody proteins resulting in reactivity to that particular substance
ix. Memory – response improves with each successive encounter x. Remembers infectious agent and can prevent later disease xi. Immunity to withstand and resist subsequent exposure to the xii. Same foreign substance is acquired
xiii. Lag phase in antibody development
xiv. Lymphocytes – recognition receptors; each B and T cell recognized only a specific microbe
xv. Others
1. Cellular: B-cells & T-cells, APCs
2. Humoral: Antibodies, Complement classic pathway, cytokines 8. Know what cytokines are and their function
a. Direct other cells of the immune system to proliferate
b. Soluble protein or peptide molecules that function as mediators of the immune response
c. Fine tune the immune system and act as critical cell activators d. Communicate between cells through the plasma
e. Increased in stored blood products with lymphocytes – can cause Febrile Txn Rxn
f. Lymphokines and monokines.
g. Regulate growth, mobility, and differentiation
h. May work together or may counter act and regulate each other.
i. Cytokines act by attaching to receptors on specific cells signaling internal cell pathways to be activated.
j. IL- Proliferation of activated Bcells and Tcells, induction of fever, acute phase proteins, bone marrow resorption
k. Cell growth, Differentiation, Chemotaxis, l. CSF- Stimulate growth of cells
m. TNF- Induce acute phase proteins, antiviral and antiparasitic activity, Activation of phagocytes
n. IFN- Antiviral, Diff of cytotoxic Tcells 9. Know categories of antibodies
i. Naturally occurring – ags are found widely in nature and have a repetitive complex nature.
1. Ubiquitous
2. Activate complement 3. RT or lower
4. Can be hemolytic if active at 37 degrees 5. ABO, Hh, Ii, Lewis, MN, P
ii. Immune – ags are unique to RBCs 1. Require AHG for detection 2. Rh, Kell, Duffy, Kidd, Ss b. Autoantibody vs Alloantibody
i. Alloantibodies: produced after transfusion, pregnancy, transplant ii. Autoantibodies: can have specificity – mostly in Rh family
iii. Must remove any autoantibody interference in order to detect underlying alloabs
10. Know types of bonds holding antigen-antibody complexes together a. Ionic: +/- charge
b. Hydrophobic: Avoid water
c. Van der Waals: (-) electron cloud + protons from another nucleus of another atom
d. Hydrogen: Hydrogen + something, incomplete transfer of energy from the hydrogen to the other atom keeps them bound
11. Know stages of hemagglutination and what affects them a. In vitro: Hemaaglutination, 2 stages:
i. Sensitization, Stage 1
1. Pairing of ag and ab happens mostly by chance – requires immunologic recognition between ag and ab
2. No visible agglutination at this point 3. Increase Serum-Cell Ratio
a. Serum increase Ab. Increases probability of collision events with the corresponding ag. Increases amt of ab available
b. Cells increase Ag. Does NOT increase reaction probability
4. Temperature
a. Temp depends on the aby different classes react at diff temp.
b. IgG – 37 degrees c. IgM - RT
d. We are most concerned with ones that react at body temp 37
5. Incubation Time
a. Allows sufficient time for reaction to occur b. Differs according to reagent and test method 6. pH
a. pH 7.0 (physiologic in body) Normal plasma pH is 6.5 to 7.5
b. Adequate for majority of abs 7. Ionic Strength
a. In physiologic saline - Na and Cl ions are attracted to charged groups of
b. Ag and Ab which hinders agglut. Decrease the ionic strength makes it easier for ag and ab to get together. ii. Lattice formation (agglutination)
1. After red cells have been sensitized with ab, random collisions between the sensitized red cells are necessary to develop crosslinkage for visible agglut to occur
2. Zeta potential:
a. Force of repulsion between two red cells in physiologic saline
b. RBC have a net neg charge in physio saline, Cations (positively charged ions) from the saline environmnent are attracted to neg charge, stable ionic cloud surrounds cells and repulsion force is increased. Therefore, cells remain apart. Distance between cells is proportional to zeta potential.
3. Ig Class
a. Because of larger pentamer shape and multivalency of IgM, easier to bridge gap and form lattice.
b. IgG abs smaller and less able to span distance between cells generated by zeta potential. IgG may attach but visible agglut may not occur
12. Know Prozone vs Postzone
a. Prozone – concentration of antibody exceeds concentration of antigen, decreases amount of RBC agglutination
b. Postzone – concentration of antigen exceeds concentration of antibody, decreases amount of agglutination
13. Know how to enhance agglutination a. Centrifugation:
i. Decreases reaction time by increasing gravitational forces on the reactants, and brings reactants closer together (closer proximity)
ii. Under centrifugation, sensitized RBCs overcome their natural repulsive effect – zeta potential
iii. Closer proximity – allows for increased ag-ab lattice formation b. Enhancement Media:
i. Reduces zeta potential – the net negative charge around RBCs that repels other cells
14. Antibodies – Know what portion binds C3 and what binds antigen a. Fab (amino) portion at the top of the antibody
i. Antigen
b. Fc (carboxy) portion at the bottom of the antibody i. C3, placental, macrophages
15. Know Unexpected vs Expected Alloantibodies a. Expected antibodies – ABO antibodies
i. all people should have unless newborn, elderly, immunosuppressed b. Unexpected – all other alloantibodies
i. People should not have these, only develop after exposure all must be investigated before transfusion
a. Polyspecific vs Monospecific
i. Polyspecific – Complement + IgG
1. Contains antibody to human IgG and to C3d 2. May also contain anti-C3b, anti-C4b, or anti-C4d ii. Monospecific – Complement or IgG
1. Contains only one antibody specificity, either IgG or Complement
b. Causes of False Negative/Positive i. False Negative
1. Neutralization of the AHG reagent can be caused by not adequately washing the cells. Also if increased volumes of plasma is used, routine washing may be inadequate.
Extraneous proteins could be mistakenly added, like covering the tube with your finger or the dropper in the reagent bottle was contaminated.
2. Interruption in testing may cause bound IgG to dissociate from the RBC and either leave too little IgG to detect or may
neutralize AHG reagent. Also agglutination of IgG coated RBC will weaken so centrifuge and read immediately.
3. Improper reagent storage: AHG reagent may lose reactivity if frozen or if bacterially contaminated. The serum can loose reactivity if excessive heat or repeated freezing and thawing occurs. Reagent red cells may lose antigen strength on storage.
4. Improper procedures: overcentrifugation may pack red cells to tightly that resuspending red cells breaks up agglutinates. Under centrifugation may not be optimal for agglutination. Failure to add the test serum, enhancement medium or AHG may cause negative test. Too heavy a red cell concentration may mask weak agglutination. Too light a suspension may be difficult to read
5. Complement: rare antibodies notably some anti-Jka, anti-Jkb, may be detected only when polyspecific AHG is used and when active complement is present.
6. Saline: Low pH of saline can decrease sensitivity. Optimal saline wash solution for most antibodies is 7.0 to 7.2. Some antibodies may require saline to be a specific temperature to retain antibody on cells.
ii. False Positive
1. Some cells may be agglutinated before washing and it does not disperse during washing. Use saline control.
2. Particles: Dust or dirt in glassware may cause clumping – not agglutination. Fibrin or precipitates in test serum may produce red cell clumps mimic agglutination.
3. Improper procedure: Over centrifugation may pack cells so tightly so they do not easily disperse and appear positive. Centrifugation of test with PEG or positively charged polymers before washing may create clumps to not disperse.
4. Cells with Positive DAT = these cells will be positive then with any AHG procedure
5. Complement: primarily C4 may bind to cells from clots or from CPDA-1 donor segments during storage at 4C and occasionally at higher temperatures. For DATs, use red cells anticoagulated with EDTA, ACD or CPD. Samples collected in tubes
containing silicone gel may have spurious complement
attachment. Complement may attach to red cells in specimens collected from infusion lines used to administer dextrose
containing solutions. Strongest reactions are seen when large bore needles are used or when sample volume is loess than 0.5ml.
c. Factors affecting test i. Temp
1. Optimal temp is 37 degrees for IgG antibodies ii. Reaction Medium
1. Albumin 2. LISS 3. PeG 4. Saline iii. Serum Cell Ratio
1. Increasing the ratio increases the sensitivity 2. Can help detect weak antibodies
iv. Incubation Time
1. LISS and PeG shortens time to 10-15 minutes 2. Saline and Albumin is 30-120 minutes
v. Washing
1. Minimum of 3 times
2. Removes protein that can neutralize AHG reagent and cause false negative reaction
3. Performed in shortest time possible to minimize elution of low affinity antibodies
4. Use saline pH 7.2-7.4 17. Primary vs Secondary Immune Response
a. Primary
i. Increased lag phase, Mainly IgM b. Secondary
i. Decreased lag phase, Mainly IgG 18. Know properties of Fc and Fab portions of antibodies
a. Fc- bind immune cells (macrophages, neutrophils). Fragment, crystallizable i. BINDS Complement
ii. Placental transfer (IgG)
b. Fab- binds antigen. Fragment, antigen binding 19. Know the terminal ends of an antibody structure
a. Amino end = Fab = Variable
b. Carboxy terminal = Fc = Non-variable
20. Humoral vs Cellular immunity and cells of each category a. Cellular: Phagocytic cells
i. B and T cells, APC’s
b. Humoral: Complement alternate pathway, cytokines
i. Antibodies, Complement classic pathway, Cytokines 21. Direct vs Indirect AHG Test
a. Direct (DAT) – Detect IgG or Complement
i. Cells sensitized in vivo (in body) – no incubation 1. Example: HDN (Hemolytic Disease of Newborn) 2. Autoimmune hemolytic anemia
3. Transfusion reaction 4. Drug mechanism
b. Indirect – Detect IgG or Complement
i. Cells sensitized in vitro (in test tube) – Incubation required ii. Two- stage procedure
iii. Patient serum and Reagent Red cells incubated first to cause antigen/antibody reaction
iv. Application:
1. Crossmatch
2. Antibody Detection and Identification 3. Titers
4. Antigen Typing 22. Principle of AHG Test
a. Antihuman globulins are obtained from immunizing animals with human globulins. These antihuman globulins are used to detect antibodies that cannot cause direct agglutination within a test system and cause visible agglutination.
b. AHG test can be used to detect IgG autoantibodies, IgG alloantibodies, and complement components. Sensitization can occur in vivo or in vitro.
c. IgM large enough to bind Ab and directly agglutinate in saline
d. IgG are called nonagglutinating because monomer structure too small, requires addition of AHG to form lattice and agglutinate.
e. The antibody to human globulin attaches and agglutinates sensitized red cells. The AHG can also bind to antibodies and complement if present in free serum. If this occurs the AHG becomes neutralized giving a false negative reaction AHG test result. Therefore, to avoid neutralization, the red cells must be washed of free unbound proteins before the addition of AHG serum. The DAT demonstrates in-vivo sensitization while the IAT shows in-vitro reactions of serum/plasma containing antibodies. An IAT is used in antibody detection, antibody identification, crossmatching and blood group phenotyping.
23. Importance of washing in AHG Test
a. Removes any unbound globulins and excess protein b. Minimum of 3 times
c. Removes protein that can neutralize AHG reagent and cause false negative reaction
d. Performed in shortest time possible to minimize elution of low affinity antibodies
e. Use saline pH 7.2-7.4
24. What are check cells and their function a. Sensitized reagent RBC
b. Validate (-) Coombs Test
c. Checks for neutralization of reagent by excess proteins 25. What can affect the washing stage of the AGH test?
26. What is the specimen of choice for the AHG test a. Purple top (EDTA)
