Basics of Immunology and Serology and Its Applications in Blood Banking, Part 2 Flashcards
FACTORS AFFECTING REACTIONS BETWEEN ANTIGENS AND ANTIBODIES
NCCCCAE PUT WIL
Nature of antibodies
Concentration of Antigens & Antibodies
Centrifugation
Chemical Reduciton
Cell Dosage
Antiglobulin Testing
Effects of Surface Charge
pH
Use of Reaction Media
Temperature
Washing
Incubation time
Location of the Antigen
are the most important antibodies in the blood banking concepts
IgM and IgG
• ______is a large pentameric molecule, making it efficient in agglutination (clumping of red blood cells) and complement activation, both critical in blood transfusion reactions.
• _____, being smaller (monomeric), can also activate complement but is better at crossing the placenta (implicated in hemolytic disease of the fetus and newborn).
IgM
IgG
IgA Deficiency Complications:
• Patients lacking IgA can produce______ antibodies if exposed to donor blood containing IgA (e.g., through transfusion).
• _____ can cause severe allergic transfusion reactions, as the recipient’s immune system recognizes IgA as foreign.
anti-IgA
Concentration of Antigens and Antibodies
The success of antigen-antibody reactions depends heavily on the antigen-antibody ratio.
For optimal results, the ratio of antigen to antibody must fall within the_____, where there is a balanced concentration of both.
Outside this zone, the reactions are incomplete or absent, leading to_____
zone of equivalence
false-negative results.
• This is the ideal concentration where antigens and antibodies bind in equal proportions.
• This balance forms large, stable antigen-antibody complexes that are visible, such as in agglutination (clumping of red blood cells).
Zone of Equivalence
• This is the ideal concentration where antigens and antibodies bind in equal proportions.
• This balance forms large, stable antigen-antibody complexes that are visible, such as in agglutination (clumping of red blood cells).
Zone of Equivalence
• Definition: A condition where there are too many antibodies compared to antigens.
• The excess antibodies saturate all antigen-binding sites, preventing cross-linking between antigens.
• As a result, visible agglutination does not occur even though antibodies are present, causing a false-negative reaction.
Prozone (Antibody Excess)
• A patient with a long-standing infection or autoimmune disease may have very high antibody levels, leading to _______.
• Clinical clue: If a patient has been sick for a long time or received antibiotics, their antigens may decrease, while antibodies dominate.
Prozone (Antibody Excess)
• Definition: A condition where there are too many antigens compared to antibodies.
• The antibodies become overwhelmed and cannot form enough antigen-antibody complexes to produce visible agglutination.
• This also leads to a false-negative reaction.
Postzone (Antigen Excess)
Example:
• A patient on the first day of fever might have low antibody levels because the immune system hasn’t had enough time to produce a significant response.
• Clinical clue: Doctors often ask, “How many days have you had the fever?” to determine whether sufficient antibodies have been generated.
Postzone (Antigen Excess)
Mechanism of Antibody Production and Its Impact
• Primary Immune Response (First Exposure):
• When the body first encounters an antigen, it takes time for_____ to recognize the antigen, activate, and produce antibodies. This delay can lead to a_____ reaction in early stages of infection.
• Secondary Immune Response (Re-exposure):
• Memory B cells respond much faster during subsequent exposures, producing antibodies more quickly. This is why postzone is less common during reinfections.
B cells
postzone
• _______ensures the correct antigen concentration in blood typing tests.
• Example: A_______ is used in blood bank testing to maintain an ideal antigen concentration relative to the antibodies in the patient sample or antisera.
• This balances the antigen-antibody ratio, promoting clear and visible agglutination reactions.
Red cell suspension
5% red cell suspension
Due to to_______, the antigen-antibody ratio is equal
• It will yield good, visible, agglutination reactions.
Red Cell Suspension
Red blood cells carry negative surface charges due to the presence of________
• It is found on______, which are integral proteins on the RBC membrane.
• These negative charges on the RBC surface repel other RBCs, preventing clumping or aggregation under normal conditions.
sialic acid residues.
glycophorins
• In the environment of the RBC, positively charged ions (like_____) are attracted to the negatively charged RBC surface.
• However, since all RBCs have the same negative surface charge, the net effect is repulsion between RBCs.
Na⁺, K⁺
• The_______ refers to the distance maintained between two RBCs due to this repulsion.
• The approximate distance between RBCs is_____.
• This distance is important because it influences whether antibodies can bridge RBCs and cause agglutination (clumping).
zeta potential
25 nm
IgM Antibody
• Structure: IgM is a pentamer (composed of 5 monomeric units), giving it a large size. Its span is______, which allows it to overcome the zeta potential distance of 25 nm.
• Function: IgM can bridge the gap between RBCs and directly cause_____
• Result: When an RBC antigen binds to IgM, clumping occurs without the need for additional reagents.
35 nm
visible agglutination.
IgG Antibody
• Structure: IgG is a monomer and much smaller compared to IgM. Its span is insufficient to overcome the 25 nm zeta potential distance.
• Function: IgG can bind to the RBC antigen (this is called______) but cannot bridge RBCs to cause visible agglutination.
• Result: No clumping occurs when IgG binds to antigens unless additional reagents are added.
sensitization
• ________, also known as _______, is a reagent that reacts with the____ region of IgG.
• This can attach to sensitized IgG antibodies (those already bound to RBC antigens) and link multiple IgG molecules, overcoming the zeta potential.
Antihuman globulin (AHG), also known as Coombs reagent
Fc
How AHG Works
1. ____binds to antigens on RBCs but does not cause agglutination due to its small size.
- AHG is added to the reaction mixture.
• AHG binds to the____ portion of IgG molecules attached to RBCs.
• This creates a bridge between IgG-coated RBCs, overcoming the____ - The result is visible agglutination, confirming the presence of IgG antibodies.
IgG
Fc
zeta potential
Why Not Use AHG for IgM?
• IgM does not need AHG because:
• Its large size…
• The Fc region of IgM is____ located and inaccessible to AHG, making AHG ineffective for IgM detection.
allows it to directly bridge RBCs and overcome the zeta potential.
centrally
• During antigen-antibody reactions, time is required for the antibody to bind to the antigen and form stable complexes.
• Incubation time ranges from______ to_____.
• For IgG, this allows sufficient sensitization of RBCs, especially when using AHG.
—————
• The presence of______ (e.g., low ionic strength solution, enzymes) can reduce the zeta potential, facilitating agglutination.
• This helps speed up antigen-antibody reactions and makes the process more efficient for blood typing and crossmatching.
30 minutes to 1 hour
reaction media
AHG Function:
• Bridges the gap that IgG cannot span due to the zeta potential.
• Binds to the Fc region of IgG to facilitate visible clumping.
(2) are used to optimize antigen-antibody interactions and overcome zeta potential challenges.
Incubation time and reaction media
_______are substances added to antigen-antibody reactions to:
1. Enhance antigen-antibody binding by reducing the incubation time.
2. Overcome challenges posed by ionic clouds, zeta potential, or structural barriers on RBCs.
Reaction media
reduces the number of ions in the environment compared to normal saline solution (NSS).
Low Ionic Strength Solution (LISS)
Commonly used in the lab as it is very cheap
Has the reduced amount of incubation
Reduces incubation time to 10 minutes only
Low lonic Strength Solution (LISS)
LISS
• In typical conditions, (2) from NSS interfere with antigen-antibody binding.
• Antibodies (via their Fab regions ) attract ions during binding, and the high ionic concentration in NSS can hinder binding.
• LISS decreases the ionic concentration, creating an environment where antigens and antibodies can interact more efficiently.
sodium (Na⁺) and chloride (Cl⁻) ions
A solution of albumin (a protein found in plasma) extracted from cows.
22% Bovine Albumin
• _____ disperses the positive charges around RBCs, which are part of the ionic cloud caused by the zeta potential.
• Dispersing these charges reduces the distance between RBCs, making it easier for antibodies to bridge the gap and cause agglutination.
22% Bovine Albumin
• Albumin extracted from cows poses a potential risk of contamination by bacteria or microorganisms, which could be transmitted to humans.
• Effect: Enhances antigen-antibody interactions by reducing the zeta potential and decreasing the space between RBCs.
22% Bovine Albumin
______ is a hydrophilic polymer that removes water molecules from the reaction.
• It creates a_____ environment, forcing the antigen and antibody molecules closer together.
• This increases the likelihood of antigen-antibody binding.
Polyethylene Glycol (PEG)
dehydrating
Mechanism of PEG
Dehydration
______are particularly useful because they modify the RBC surface, improving antigen-antibody reactions.
They are categorized as proteolytic ____ because they break down proteins.
Enzymes
Enzymes
• Function:
Breaks down protein present in the
surface of the red cell. Either…
- To reduce zeta potential
- For antibodies to detect small antigens near the RBC membrane
Why Use Enzymes?
1. Reduce Zeta Potential:
• ______residues on glycophorins are cleaved, reducing the RBC surface charge and minimizing repulsion between RBCs.
- Expose Hidden Antigens:
• Some antigens are buried beneath the RBC surface or within a high-protein layer.
• Enzymes break down these____, exposing the antigens to antibodies for binding.
Sialic acid
proteins
Bromelain
Conc.
pH
Function
0.5%
pH 5.5
Works in an acidic environment to reduce zeta potential and facilitate agglutination.
Ficin
Conc.
pH
Function
0.1%
pH 7.3
Works in neutral pH to enhance antigen detection and antibody binding.
Trypsin
Conc.
pH
Function
0.1%
pH 7.3
Similar to ficin, used at neutral pH to enhance antigen-antibody interactions.
Papain
Conc.
pH
Function
0.1%
pH 6.5
Functions in a slightly acidic environment but may cause hemolysis if the solution is too acidic.
Works in an acidic environment to reduce zeta potential and facilitate agglutination.
Bromelain
Works in neutral pH to enhance antigen detection and antibody binding.
Ficin
Similar to ficin, used at neutral pH to enhance antigen-antibody interactions.
Trypsin
Functions in a slightly acidic environment but may cause hemolysis if the solution is too acidic.
Papain
Certain antibodies like Anti-M react better in slightly acidic conditions.
• ______is used for confirmation tests in some cases where other antibodies may fail to detect specific antigens.
Acidic pH (6.5)
Anti-M
Most antigen-antibody reactions occur optimally in this range since it’s close to the body’s natural pH.
Neutral pH
Temperature
Antibodies are classified as cold-reactive (_____) or warm-reactive (____) based on the temperature at which they bind antigens effectively.
IgM
IgG
IgM
Optimal temp
IgM antibodies are large pentamers that work best in cooler conditions, such as refrigeration or room temperature.
4°C (cold) to 22-24°C
IgG
Optimal temp.
IgG antibodies are smaller and optimized to function at normal body temperature.
37°C (body temperature)
• The time required for antigens and antibodies to interact and bind in optimal conditions.
incubation time
Saline (NSS)
Incubation time??
NSS is a neutral diluent, so antigen-antibody interactions occur naturally but slowly.
30-60 minutes
LISS/Albumin/PEG
Incubation time???
These media enhance binding by altering ionic strength, reducing zeta potential, or dehydrating the environment.
10-15 minutes
removes unbound antibodies, impurities, or any other substances that may interfere with testing.
Only RBCs should remain for testing.
Washing
Steps for Washing:
- Mix RBCs with saline and centrifuge.
- Remove the supernatant (impurities and NSS).
- Repeat until only clean RBCs remain.
Washing
Key Considerations:
• _______: Ensures no impurities remain that can neutralize antibodies or cause false agglutination.
• _______:
• If washing is too slow, dilution of the RBC suspension occurs, leading to a false negative result.
• If washing is too fast, hemolysis may occur, destroying RBCs and invalidating the test.
Thorough Washing
Rapid Washing
Critical for AHG Testing:
• AHG reacts with human IgG bound to RBCs. If unbound IgG remains in the suspension due to improper_____, it will interfere with the reaction, causing false negatives.
washing
Centrifugation enhances agglutination by forcing RBCs closer together, allowing antigen-antibody complexes to form and become visible as clumping.
• Optimal Timing:
• ________: Sufficient to promote RBC settling without damaging cells.
Avoid Overcentrifugation:
No need for 5 minutes
1 minute is way too much
5-10 seconds
is used to remove interference from IgM antibodies and ensure accurate results, especially when performing tests at room temperature.
Chemical reduction
Why IgM Causes Interference
• At Room Temperature:
• IgM is reactive at room temperature (_____).
• When performing blood typing or antibody screening, IgM may cause unwanted reactions that interfere with test results.
• Problem:
• IgM is large and can cause clumping of red blood cells (agglutination), but it’s not always useful for detecting the antigens we need to identify.
• To avoid false results, IgM needs to be removed or broken down, so only the smaller____ antibodies remain for the test.
22-24°C
IgG Ab
Chemical reduction
IgM should be_____ or we should get IgM through____ so IgG would be left
broken down
absorption
Chemical breakdown of IgM into monomeric units
Reducing agents (Chemical Reduction
______ : Breaks the disulfide bonds that hold the IgM together.
______: Also used to break disulfide bonds in IgM.
______ : A combination of DTT and an enzyme (such as bromelin/ papain) that helps reduce disulfide bonds, especially in IgG.
Dithiothreitol (DTT)
β-mercaptoethanol
ZZAP (IgG)
ZZAP is a combination of_____ and _____
will reduce the disulfide bonds in the hinge region of the IgG
This extends the Fab portion of our IgG so that it further spreads; this wider span allows IgG to attach from one red cell to another
This is mainly possible if potentiators are added.
Zeta potential reduced from potentiators + IgG hinge region reduced
= higher propensity to attach to red cells
dithiothreitol and an enzyme mostly bromelin
Is used to detect non-agglutinating antibodies, particularly IgG antibodies or complement components like C3d, that are bound to red blood cells (RBCs).
Antiglobulin testing
There are two main types of antiglobulin tests:
Direct Antiglobulin Test (DAT)
Indirect Antiglobulin Test (IAT)
• Purpose: To detect antibodies (mainly IgG) or complement components (like C3d) that are already attached to RBCs in vivo.
Direct Antiglobulin Test (DAT)
Process:
• Red blood cells are already coated with IgG antibodies or complement components (C3d).
• The test detects sensitization of RBCs to these antibodies or complement components.
• AHG (Anti-Human Globulin) is added to the sample. AHG specifically binds to the Fc portion of IgG antibodies or C3d.
• Agglutination occurs because AHG links the IgG or C3d on the RBC surface, causing clumping.
Direct Antiglobulin Test (DAT)
DAT
• Sample Used:
Red blood cells (not plasma).
DAT
• Key Point: The test is used to detect_________ (inside the body), where antibodies or complement have already bound.
pre-sensitized RBCs in vivo
• Purpose: To detect antibodies that are not yet attached to RBCs, but will be sensitized in vitro (in the laboratory).
Indirect Antiglobulin Test (IAT)
• Process:
• Phase 1: Sensitization: RBCs are mixed with a sample that contains IgG antibodies. If the antibodies are present, they bind to antigens on the red cell surface.
• Phase 2: Addition of AHG: After sensitization, AHG is added. If the IgG antibody has attached to the RBCs, agglutination will occur.
Indirect Antiglobulin Test (IAT)
IAT
• Sample Used:
This test can use either red cells or plasma.
• Key Point: The test detects antibodies not naturally present on RBCs, but those that the lab can introduce.
IAT
• If what you want to find out is a certain ANTIGEN what will serve as your sample is the______ and what will serve as your reagent is_____
• If what you want to find out is a certain ANTIBODY what will serve as your sample is the_____ and what will serve as your reagent is_____
RED CELL; PLASMA
PLASMA; RED CELL
When dealing with antiglobulin testing, the antibody involved is always____.
IgG
• Contains antibodies to human IgG and human C3d.
• Purpose: Screening test to detect both IgG antibodies and complement on RBCs.
Polyspecific AHG
Polyspecific AHG:
Advantage
Disadvantage
• Advantage: Can detect complement-dependent antibodies (e.g., anti-Jka).
• Disadvantage: May lead to nuisance positives (false positives), since it can react with both IgG and C3d.
• Contains antibodies to only IgG or only C3d (separate bottles).
• Purpose: Confirmatory test to confirm which component (IgG or C3d) is bound to the RBC.
Monospecific AHG
Monospecific AHG
Advantage
Disadvantage
• Advantage: Fewer nuisance positives.
• Disadvantage: May miss important antibodies that polyspecific AHG could detect.
Note: It is not recommended to test directly with________, because if the polyspecific AHG is negative, the AHG test will likely also fail, leading to unnecessary use of reagents.
monospecific AHG
• Content: IgG coated red cells
• Purpose: To confirm negative result in AHG (+) Reaction: Visible agglutination (The sample is truly negative)
Coombs Check Cells
Coombs Check Cells
• Content:
• Purpose:
IgG coated red cells
To confirm negative result in AHG (+) Reaction: Visible agglutination (The sample is truly negative)
• ______is used to detect sensitization of RBCs by IgG or complement in vivo.
• _____is used to test for antibodies that can be sensitized to RBCs in vitro.
• ______reacts with both IgG and C3d, used for screening.
• ______reacts with either IgG or C3d, used for confirmatory tests.
• _______are used to confirm a negative result by ensuring the AHG has worked correctly.
DAT
IAT
Polyspecific AHG
Monospecific AHG
Coombs check cells
refers to the number of antigens present on the surface of red blood cells, which depends on the genes inherited from each parent.
Cell dosage
• When you inherit different alleles for an antigen (e.g., AO or BO), there will be less antigen production, resulting in lower reactivity.
• Example: • AO → A antigen (A antigen is present in a lower amount) • AB → A and B antigens (Both A and B are present in moderate amounts) • BO → B antigen (B antigen is present in a lower amount)
Heterozygous Production
• When you inherit the same allele from both parents (e.g., AA or BB), more antigen is produced, resulting in higher reactivity.
• Example: • AA → A antigen (Higher amount of A antigen) • BB → B antigen (Higher amount of B antigen)
Homozygous Production
• _______individuals will have less antigen and therefore less reactivity.
• _______individuals will have more antigen and therefore more reactivity.
Heterozygous
Homozygous
Location of the Antigen
• Some antigens are located in the red cell membrane, such as the_____ and____ (which house antigens like MNSs).
• The location of these antigens is important because antibodies need to be able to reach the antigen to bind with it. If glycophorins (proteins) cover the antigen, it may be difficult for antibodies to attach.
• _______can be used to remove the glycophorins, allowing antibodies to better access the antigen.
Rh antigen; glycophorins
Proteolytic enzymes
- most often not used
Needs to be considered because when RBC bursts/ dies, we now have no antigen
A good sign in immuno because a
complement was activated.
Reasons:
(2)
Hemolysis
Over centrifugation; Wrong diluent
Is caused by increased proteins in the
plasma (CRP) → Due to infection or
inflammation → results to decreased zeta potential → RBCs will now clump/stack like coIns
Reason: …
Solution: …
Rouleaux formation
cells were not washed prior to
making red cell suspension
If it occurs in vivo = wash and
the Rouleaux formation is solved
• In vivo sensitization or presence of cold reacting (IgM) antibodies
Or the patient has autoantibodies
Sample problem
Try to check the lot numbers and the
expiration dates before performing
the procedure
Always make sure to have the reagents in ROOM TEMP before using it.
Reagents used
____
Make sure pipette and centrifuges are
calibrated
____
Question of skill, skipped or omitted some steps
• Equipment used
• Technical Problems
Special Techniques in Blood Banking
- Enzyme Treatment
- Elution of Antibodies from the surface of RBCs
- Adsorption
- Neutralization
- Saline Replacement Technique
• Can be done in the patient’s sample or could be utilized as the procedure to make your reagent red cells
• Enzymes remove very long antibodies such as your daffy A and daffy B and MNS
Enzyme Treatment
Disassociating Abs from RBC membranes
When we centrifuge, antibodies attached to RBCs will dissociate and be displaced into the supernatant
Avoid putting the centrifuge in the same table as the red cell suspension is placed because it causes the table to shake and eluate the suspension
• Supernatant with antibodies is called the Eluent
Eluent can be used for antibody screening or testing for the detection of antibodies.
Elution of Antibodies from the surface of RBCs
Removal of Ab from serum by combining serum with appropriate RBCs
If there is an unwanted antibody it will be adsorb using a RED CELL that contains a particular antigen
So what will be left is the antibody of interest
Useful if the patient has both autoantibody and an antibody against the antigen of another individual
Adsorption
Uses soluble Ag to inhibit reactivity of some Abs in testing
Just like adsorption, but with the use of a SOLUBLE SUBSTANCE
The unwanted antibody will form a
precipitate with antigen
Neutralization
• Removing the serum and replacing with saline dilutes the interfering proteins so that rouleaux formation is prevented
Saline Replacement Technique
Proteolytic substances
Modifies RBC membrane by reducing sialic acid residues reducing surface charge, splitting polypeptide chains and exposing or destroying antigens
• E.g. papain, bromelin, ficin, trypsin
Enzyme Treatment
