Applications of immunology and

Serology in Blood Banking

- By Ms. Candelari  ♥
Recall: Immune System
Branches of Immune Response: Innate
vs Adaptive

Innate Immunity Adaptive

Immunity
First line of defense Second line of defense Third line of defense

 Intact skin  Phagocytes  Specialized

 Mucous membranes and their
secretions
 Normal microbiota
(neutrophils,eosinophils, dendritic lymphocytes: T cells
cells and macrophage) and B cells
 Inflammation  Antibodies
 Fever
 Antimicrobial subs
Complement Activation Pathways
Patient’s Immune Response Against
Incompatible Blood
 Characteristics of Antigens

“All immunogens are antigens,

but not all antigens are immunogens”
• Antigen – capable of reacting with an antibody
• Immunogen – initiates an immune response
• Hapten – small molecules that do not elicit an
immune response on their own; needs a carrier
 Antigen Properties that Influence
Immune Response
1. Foreignness
2. Size
3. Complexity
4. Accessibility
5. Digestibility
6. Stability
7. Conformation
8. Chemical composition
 RBC Antigen Compositions
• Rbc antigen are very diverse in structure and
composition and may be:
• Glycoprotein
 Rh
M
N
• Glycolipid
 ABH
 Lewis
 Li
 P
• Protein
 HLA
 Antibody at a
Glance

• Valency – number of antigen binding sites

• Epitope – antigenic determinant
• Paratope – aka. antigen binding site;
clusters of complementarity-determining
regions (CDRs) in the hypervariable
regions
 Immunoglobulin Gamma
• Warm reacting antibody
• 37 degrees Celsius
• Can cross placenta
- IgG1 (+), IgG2 (+)
- IgG3 (+), IgG4 (+)
• Can fix complement though not
as efficient as IgM (needs 2 CH2):
- IgG1 (++), IgG2 (+)
- IgG3 (+++), IgG4 (0)
• Most clinically significant
antibodies (affinity to antigen)
• Can cause anemia and
transfusion reactions
Macrophage-binding region
 Immunoglobulin Mu
• Cold reacting antibody
• 4 degrees Celsius
• Room temperature
• ABO Blood group system
• Naturally occurring antibody
• Primarily testing program: IgM
antibodies interfere with
detection of clinically significant
IgG antibodies by masking their
reactivity
• Remedy to take out IgM: B2-
mercaptoethanol (2-ME) or
dithiothreitol (DTT)
 Characteristics of Blood Group
Antibodies
1. Polyclonal vs Monoclonal Antibodies
2. Naturally occurring vs Immune Antibodies
3. Alloantibodies vs Autoantibodies
 Polyclonal vs Monoclonal Antibodies
• Antisera – reagent antibodies
• Polyclonal/serum antibodies –
produced in response to a single
antigen with more than 1 epitope
• Monoclonal antibody – antibody
formed by a single type of B cell,
clonally expanded, against an antigen
of a single type of epitope.
 Naturally Occurring vs
Immune Antibodies
• Naturally occurring antibodies –
aka. ISOAGGLUTININS, are Most common NOA
antibodies found in the serum of • ABH
• Hh
patients who have never been • Li
exposed to that antigen (mostly • MN
igM) • Lewis
• P
• Immune antibodies – antibodies Most common IA
found in the serum of patients who • Rh
have been exposed to antigen prior • Kell
transfusion or pregnancy (mostly • Duffy
• Kidd
IgG) • Ss
 Unexpected Antibodies!

• Sensitized patients –patients who were previously exposed to antigen.

 Alloantibodies vs Autoantibodies
Two unexpected red cell antibodies:
• Alloantibodies – produced after exposure to
genetically different, or non-self antigens such
as transfusion (RBCs,WBCs, or even platelets
not present in patient)
• Autoantibodies – produced in response to
self-antigens and can react at both warm and
cold temperatures
 Characteristics of Antigen-Antibody
reactions
1. Intermolecular binding forces
- Hydrogen bonds
- Electrostatic forces
- Van der Waals forces
- Hydrophilic bonds
- Hydrophobic bonds
2. Antibody properties
- Affinity vs Avidity
- Specific reactions
- Cross-reaction
- No reaction
3. Host factors
4. Tolerance
 Factors that influence agglutination
reactions
1. Charge
2. Centrifugation
3. Antigen-antibody ratio
4. Effect of pH
5. Temperature
6. Immunoglobulin type
7. Enhancement media
8. Protein media
9. Low ionic strength solution (LISS)
10. Proteolytic enzymes
 Centrifugation
• Decrease reaction time by increasing
gravitational forces and bringing reactants
close together
• Sensitized RBCs overcome their natural
repulsive effect (ZETA POTENTIAL) for each
other and agglutinate more efficiently
 Antigen-Antibody Ratio

• Prozone – excess of unbound antibodies causing false negative

reaction
• Postzone effect – surplus of antigen causing false negative reaction
• Zone of equivalence – number of binding sites of multivalent antigen
and antibody are approximately equal
 Grading Agglutination Reactions

• To standardized element of subjectivity among Blood Bank

personnel performing the testing
• May be performed in test tubes, microplate wells, microtubules
filled with gel particles
• Conventional grading for the tube system uses a 0 to 4+ scale
 Effect of pH
• pH (power/potential of hydrogen) – figure
expressing the alkalinity or acidity of a solution.
• Ideal pH of a test system: pH- 6.5-7.5
• Normal blood pH is tightly regulated at 7.35-7.45
• Except these antibodies which react best at pH<6.5:
- Anti-M
- Pr(Sp1)
• Acidification of test serum may aid in distinguishing
anti-M and anti-Pr(Sp1) antibodies from others
 Temperature
• Immediate spin (IS) phase:
- Ambient room temp.
- <22 degrees Celsius
- IgM antibodies
• 37 degrees incubation phase
- IgG antibodies
• Antihuman globulin (AHG) phase:
- IgG antibodies
 Immunoglobulin Type
• Differences in the nature of reactivity between
igM and igG antibodies require different
serologic systems to optimally detect both
classes of clinically significant antibodies
Immediate Spin phase (IS) Antiglobulin phase / 37C (IgG)
• A,B,H • Ss
• I,I • Kell (Kk,Js(a),Js(b),Kp(a),Kp(b))
• M,N • Rh (DCEce)
• Lewis (Le(a),Le(b)) • Lutheran (Lu(b))
• Lutheran (Lu(a)) • Duffy (Fy(a),Fy(b))
• P • Kidd (Jk(a),Jk(b))
 Enhancement Media
• Sialic acid on RBC membrane cause
RBCs to repel each other since most
acids have a negative charge and
therefore creates this large zone of
negative charge around cell
• Zeta potential – expression of the
difference in the electrostatic charges
at the RBC surface and the
surrounding cations (antibodies)
• Reduces the zeta potential of RBC
membranes:
1. Saline Media
2. Protein Media
3. Low Ionic Strength Solution Media
4. Proteolytic Enzymes
 1. Protein Media
• Colloidal substances – a type of clear
solution that contains particles
permanently suspended in solution; can
be charged or neutral
- Examples:
1. 22% Albumin
2. Polyethylene glycol (PEG)
3. Polybrene
4. Polyvinylpyrollidone (PVP)
5. protamine
• Crystalloids – small, highly soluble
molecules that are easily dialyzed
- Examples:
1.Glucose
 2. Low ionic Strength Solution Media
• Also known as Low Salt Media
• Generally contain 0.2% Sodium Chloride
• Increase rate of antibody uptake during
sensitization
• Compared with protein
potentionators/media, LISS decrease
incubation time (from 30-60 minutes to
5-10 minutes)
• However may result in false-positive
reactions and may require testing to be
repeated with albumin
 3. Proteolytic Enzymes
Enhanced Antibody reactivity Antiglobulin phase / 37C (IgG)
• Rh • Fy(a)
• Kidd • Fy(b)
• P1 • M
• I • N
• Lewis • S

• Enzymes – protein molecules that function by altering reaction

conditions and brining about changes in other molecules without
being changed themselves
• Substrate – molecule that enzymes react with
• Proteolytic enzymes – target protein molecules
- Ficin (isolated from fig plants)
- Papain( from papaya)
- Trypsin (from pig stomach)
- Bromelin (from pineapple)
• Enzymes release sialic acid from RBC membrane thus decreasing
zeta potential and negative charges
 So what have we learned so far?
• That having a strong foundation in immunosero
can help us understand the principles in blood
banking and transfusion medicine
• That antibodies have different characteristics thus
it is very crucial for blood bank personnel to
detect and investigate them properly
• That antigens have different properties which
influence antigen-antibody reactions
• That technology has allowed more efficient work
in the blood bank that air in testing and faster
response to patient care