OVERVIEW OF IMPORTANT

IMMUNOLOGIC PRINCIPLES IN BLOOD

BANKING LECTURE 2021 – 2022
IMMUNOHEMATOLOGY 2nd Semester
Instructor: Joseph Joy Banzon, RMT, MPH WEEK 3 IMHM321
Date: February 28, 2022

TOPIC OUTLINE ➢ Antigens and Immunogens are same term, but in the
I. Introduction definition; antigens bind with the antibody, Immunogen
II. Antigen
III. Blood Group Specific Antigen bind with the antibody but only immunogen can stimulates
IV. Blood Transfusion the production of antibodies
V. Antibodies ➢ Not all antigens are immunogens but all immunogens
VI. Complement System are antigens
VII. Interpreting Hemolysis Reactions in Blood
Bank ➢ The antigens must be capable of achieving the following
VIII. Antigen-antibody Reaction factors.
• Intermolecular Forces that bind AG and AB
together FACTORS AFFECTING IMMUNOGENECITY
IX. Agglutination
• Steps in Agglutination 1. Foreigness - the substance must be foreign; if the
• Factors Affecting Sensitization antigen is not foreign there will be no antibody
• Factors Affecting Lattice Formation production and the more foreign the antigen is the
more immunogenic
➢ We will not produce antibody against a not foreign material
especially self antigens
INTRODUCTION ➢ Whenever produced antibodies destroy self antigen>
theres a problem> autoimmunedisease
⚫ IMMUNOLOGY - Immune system - recognize and sipose ➢ Antibody will not produce on self antigen
foreign antigens
➢ Foreign antigens refers to antigens of microorganism like 2. Chemical Complexity - the more complex the
viral antigens and bacterial antigens substance the more immunogenic
➢ As human being, human have antigens in the human ➢ The most immunogenic are the ones that are made up of
body it would be the blood group antigen or the red cell proteins, carbohydrates, glycoprotein, glycolipid, lipid and
antigen nucleic acid
➢ Red Cell Antigen - dictates and control what the blood ➢ Lipids and Nucleic Acid are poor immunogens therefore
type will be the antigens of bacteria, viruses are made up of any
➢ Even though humans have own self antigens, but during following substances
blood transfusion or if ever the donors blood type is not ➢ Majority in the microbes are made up of protein
the same with the patients blood type, the patient may ➢ Antigens in the red cell that dictates blood type are made
exposed to a foreign antigen up of protein, carbohydrates, glycoprotein and glycolipid
⚫ IMMUNOHEMATOLOGY - application of principles of ➢ For example, the RH antigen that dictates if RH Positive
immunology to study of blood group specific antigens and or RH Negative is made up of pure proteins therefore the
antibodies RH Antigens are highly immunogenic, there is no way that
➢ The principle of the immunology pertains to antigen can transfused an RH Positive to RH Negative person
antibody reaction and read concept that will be used in ➢ And if the RH negative person exposed to RH positive
studying red cell antigens that dictates the blood type and blood, expect the patient instantly produced antibody
corresponding antibodies against the corresponding RH antigen
⚫ Blood Banking- science that deal with the collection, ➢ For example, ABO Antigens of the ABO Blood group
preservation, processing, storage and disposal of donors system are made up of other glycolipid and glycoprotein
blood with strict adherence to quality control and as complex materials or substances they can be acting as
quality assurance immunogen therefore they are capable of stimulating
antibody production in an easy manner
➢ The donors blood is regarded as drug or medicine
➢ The donors blood is transfused to patients whom may
3. High Molecular Weight
need it
➢ The size or the molecular weight should be high
➢ One of the corrective way that the doctor to addressed
➢ The size should be more than 10,000 Daltons
the diseases or the condition of the patient
➢ The higher the molecular weight the more immunogenic
➢ Just like other materials, food that are regulated must 4. Digestability
ensure giving nothing but quality blood supply for patients
➢ The antigens or the substance must be digestable
➢ It must be capable of broken down into smaller pieces
ANTIGEN because antigens bind with the antibody or the T
lymphocyte, and the lymphocyte cannot bind with the
⚫ ANTIGENS - substances that binds with antibody; antigen not unless they are processed and digested by
sunstances that can bind with T- Lymphocyte APC’s and presented along with MHC
⚫ Also called as: Immunogens - can stimulate the production ➢ If cannot be broken down into smaller pieces it cannot be
of antibodies processed with the APC’s the antigen
➢ If there is no APC it cannot bind with the antigen

B. LEGASTE & M. VALENCIA – TRANSCRIBERS

 5. Dosage and Route of Administration GRADING AGGLUTINATION RECATION
➢ Intravenous route is the fastest way for an antigen to
cause antibody reaction GRADE CLUMPS SUPERNATANT/
➢ Blood transfusion is done intravenously BACKGROUND
➢ The transfused blood contained a foreign material the 0 (NEGATIVE) No Clumps/ No Red, Turbid
exposure to the corresponding b cell that produced Agglutination
antibodies are also direct hence the fastest way to +/- (Trace or Very Tiny Clumps Red, Turbid
immunogen to cause antibody production is that when the weakly positive)
antigen entered human body via intravenous route of mf
(Mixed filled
agglutination -
6. Accessibility of Reactive Sites presence of very
➢ Antigen must have available and reactive sites to be able tiny cumps of
to bind with the corresponding antibody or T cell RBC floating in a
sea of free red
*not all antigens are antigen, they are must 1st capable of blood cells)
having that characteristics before be able to cause the 1+ Small Clumps Red, Turbid
stimulation of the production of the antibodies
2+ Medium Sized Clear
3+ Several Large Clear
BLOOD GROUP SPECIFIC ANTIGEN Clumps
4+ One Large Clear
Clumps
BLOOD GROUP SPECIFIC ANTIGENS/ RBC ANTIGENS
Blood Group specific antigen that dictates what are Blood ➢ Grading ranges from 0 to 4+
type is. ➢ Negative - no antigen reaction had occur
➢ If your red blood cell is A antigen, Type A ➢ From trace to 4+ is positive
➢ If your red blood cell has A and B antigen, Type AB ➢ There has been antigen antibody reaction had taken
➢ The presence of specific antigen in red blood cell will tell place
what the blood type is. ➢ TRACE- has weak reaction, there are clumps but very
tiny in red background and it may difficult to find in
⚫ NOTE: As of latest editions of Blood Banking Books, observing in naked eye, LESSON: if walang makitang
there are >250 RBC Antigens that are existing and are clumps do not report it as 0, do not declared it
group into 36 Blood Group system immediately as negative it should be looked under the
➢ We have different blood type that make us unique microscope, floating clumps in free red blood cell > trace
➢ In blood transfusion the 2 most important blood type is or mixed filled agglutination
ABO Blood group and RH Blood group
➢ Since our blood type dictates what antigen present in the
red blood cell, if you want to know the blood type, detect
what type of antigen present in the patients red cell

RBC Antigens are detected through: DIRECT

HEMAGGLUTINATION REACTIONS (Principle of Blood
typing Procedures)

3 METHODS

1. Slide Method - results cannot be graded; Positive or

Negative
➢ Not sensitive for doing the blood typing
➢ Results cannot be graded
➢ Clumps> Positive No Clumps> Negative

2. Tube Method - more specific than slide method

➢ Grading the agglutination reaction means determining the
strength of agglutination reactions 3. Gel Method
➢ Not just simply positive or negative ➢ Sa loob ng plastic tube may clear gel
➢ Grading reaction is possible beginning with the tube (POLYACRYLAMIDE GEL)
method ➢ Papatakan ng patient red cell, sa gel naka mixed na ang
anti-sera
➢ Centrifuge the gel card

B. LEGASTE & M. VALENCIA – TRANSCRIBERS

 ⚫ Platelet Refractoriness - unresponsiveness to platelet
transfusion because of the ANTI-HLA that binds and
destroys HLA on platelet’s surface
➢ Usual cause due to multiple platelet transfusion (suki sa
pagtransfused ng platelet ang pasyente)

➢ HLA matching are not routinely test during blood

transfusion
➢ If ever the donor possess or different HLA antigen and
that patient exposed to that foreign antigen expect the
patient to produce antibody on platelets surface
➢ Ones the patient already produced anti HLA antibody,
expect that the HLA antibody to destroy the Platelets that
be transfused further
➢ Blood transfusion can save lives but because of multiple
ADVANTAGE blood transfusion it may risk the life of the patient
1. Grading reactions are objective ➢ Platelet Refractoriness> Pag madalas and Blood
➢ Gel Technology transfusion
2. Small amount of sample to be used
3. Results are stable for up to 3 days - can review the results
ANTIBODIES
after 3 days

DISADVATAGE ⚫ Antibodies - Glycoproteins that are produced in response

1. Costly/ expensive - special designed centrifuged, Gel cards to antigenic stimulation
⚫ Also known as : Immunoglobulin; part of gamma Globulin
➢ Discovered by Dr. YVES LAPIERRE in Lyon, France fraction of seerum proteins
made the lives of blood bank easier ⚫ Basic Functional and structural unit is: MONOMER- Y
⚫ Solid Phase Red Cell Adherence (SPRCA): Discovered shaped molecule and tetrapeptide made up of 2 heavy
by Plapp and coworkers in 1984 chain and 2 light chains
⚫ Human Leukocyte Antigen (HLA): encoded by MHC ➢ Middle portion: “hinge region” flexible region and high
Genes located on chromosome 6 amount of proline
⚫ HLA are present not only on leukocyte but on all
nucleated cells (part of self antigen) 5 CLASSES (Arranged according to decreasing concentration
in the serum)
• IgG
BLOOD TRANSFUSION • IgA
• IgM
• IgD
⚫ Whole Blood - Red Blood Cell, White Blood Cell,
Platelets, Plasma • IgE
➢ Rbc has no nucleus, then no HLA, WBC has nucleus, ➢ IgG- most abundant
then they have HLA, Platelet has no nucleus but they are ➢ IgE least abundant
fragments of megakaryocyte and megakaryocyte has ➢ IgE increases during allergic reaction and parasitic
nucleus, platelet expressed HLA infection
➢ During Blood transfusion, aside from red blood cell, may ⚫ Antibody classes most commonly encountered in the
be exposed to foreign HLA antigen that are present on blood bank are: IgG, IgM and IgA
the membrane in WBC and platelets ➢ IgA - caused of anaphylactic transfusion reaction
➢ And in doing blood transfusion, done through blood typing
and cross-matching 2 Fragments:
➢ During cross- matching, we only matched the red cell Fab (Antigen Binding Fragment)
antigen compatibility between the patient and donor Fc (crystallizable Fragments)
➢ HLA Matching is not done routinely during blood ➢ In one monomer, there are 2 Fab, there are 2 antigen can
transfusion monomer bind, 2 binding site, 2 valence
➢ Should you given a whole blood, maybe exposed to HLA ➢ Valence - number of binding sites
antigen in donor wbc and platelet and may react against ➢ IgM is pentamer, 10 valence of antigen
that if ythe donor have the different type of HLA compare ➢ 1 Fc for every monomer
to you
➢ There are different transfused reactions, it is not limited to Regions: Variable, constant
Hemolytic transfusion reaction that occur when the red
cell blood type is different between the patient and the Fab/ variable region - binds with the antigen
donor Fc/ constant region - binds with the host cell phagocyte and
➢ But other forms and types of transfusion reaction may be complements
due to incompatibility between HLA donors HLA to wbc
Function of antibodies
and platelets
1. Binding Antigen
➢ Example of transfusion reaction incompatibility > platelet
2. Opsonization
refractoriness
3. Complement Fixation and Activation

B. LEGASTE & M. VALENCIA – TRANSCRIBERS

 ➢ What’s the result of complement activation?
Point of IgM (pentamer)- IgG (monomer) ➢ Lysis of the cell.
Comparison star ➢ Therefore, kapag nagb-blood typing (nagmix ng red
shaped/crab- cells’ patient and anti-sera) and nagbind ‘yung
shaped) Antibody ng anti-sera, mac-clump ang RBC.
1. Heavy chain mu gamma ➢ Kapag nagbind ang Antibody sa RBC, are you also
2. Light chain Kappa/lambda Kappa/lambda forming immune complex? Yes. Can this immune
3.J-chain complex trigger compliment activation? Yes.
(connects YES NO ➢ Kapag sa blood typing, kapag malakas ang reaction,
monomeric units
malakas ang Antibody hindi lang agglutination ang
of polymer)
pwede makita; pwede ring from agglutination leads
4. Molecular 900,000 daltons 150,000 daltons to compliment activation and therefore hemolysis
Weight (19s- (7s) ➢ Hemolysis is also regarded as a POSITIVE RESULT
sedimentation in Blood Banking Tests. This is due to the fact that
constant)
some antigen-antibody reactions proceeds to
5.Valence
complement activation; hindi lang agglutination ang
(number of 10 2
binding sites) tinitignan para masabing positive yung test na
ginagawa sa blood bank.
6. % in Serum 6-10% 75-80%
7. Serum half-life 5-10 days 23-25 days Remember: Hindi porket blood banking ay palaging
8. Crosses the NO YES (cause HDN) agglutination.
placenta ➢ Pwede bang gumamit ng hemolyzed sample sa
9. Activates the blood banking? Why? No, kasi magiging false-
classical YES (very YES (IgG3, IgG1, positive. So, you have to reject the specimen.
pathway of efficient) IgG2) (IgG4
complement) cannot Compliment-fixing antibody
10. Clearance of Intravascular- Extravascular- FUNCTIONS
red cell within the blood outside the blood 1. Enhance phagocytosis
vessel vessel
2. Enhance antibody function
*spleen
3. Clears immune complexes in blood
4. Kill microbes by cell lysis
11. Detection in Immediate Spin AHG Phase (Anti- 5. Cause the increase in vascular permeability
blood bank tests Phase (IS) - human globulin)/ 6. Enhances platelet aggregation
Room Coomb's Test 7. Enhances smooth muscle contraction
Temperature
8. Ais in viral neutralization

CHEMOTAXIS- Migration of phagocytosis toward the site of

➢ Culling - process when old damaged senescent rbc are
infection on inflammation.
removed by the red pulp of the spleen
➢ IgM- Cold Antibody, Can react on Room Temperature C3d: Degradation product of C3b
➢ IgG- Warm Antibody, cannot be detected on Room C4d: Degradation product of C4b
Temperature, on 37 degree celcius IgG can react, ➢ Nagbind ‘yung antibody sa RBC>> nag-form ng
requires
immune complex>> na-activate ‘yung isang
incubation
complement system>> kung ano-anong complement
IgM- Pentamer, can bind up to 10 Red blood cell, 1 molecule of
IgM is enough to cause clumping and agglutination of red fragments ang na-produce like C3b; maaring ma-
➢ blood cell deposit sa surface ng RBC ‘yung C3b. Then ‘yung
➢ IgG- Monomer, can bind only 2 Rbc, not enough to cause C3b kapag nagdegrade, magiging C3d.
clumping and agglutination reaction; aside from ➢ For example, nag-test ng RBC, nag-mix ng anti-C3d
incubating into 37 degree celcius, IgG still needs AHG antibody-reagent sa RBC, then nag-positive dahil may
Phase/ Coomb’s Test C3d na naka-deposit sa surface ng RBC.
➢ AHG- responsible for clumping IgG coated RBC, without ➢ Kapag may C3d compliment>> complement systems
adding AHG, IgG cannot be detected in the blood bank are activated because nagkaroon ng positive antigen-
➢ AHG Test/ Coombs Test - for the detection of IgG antibody reaction (o diba boom panes char)

COMPLEMENT SYSTEM Opsonin: C3b

Chemotaxin: C5a
• it completes or complement the action of antibodies
➢ Substance that can cause chemotaxis
Recap: Meron kang antibody nagbind sa antigen kapag hindi
CHEMOTAXIS- Migration of phagocytosis toward the site of
kaya ni Antibody yung Antigen >>Antibody-Antigen complex
infection on inflammation.
will trigger the activation of the compliment system

B. LEGASTE & M. VALENCIA – TRANSCRIBERS

 INTERPRETING HEMOLYSIS REACTIONS IN BLOOD BANK 4. Van der Waals Forces- attraction of the electron
clouds of the antigen and antibody
• Complete Hemolysis- Red supernatant. No cell
button. AGGLUTINATION
- All of RBCs are hemolyzed.
2 STEPS IN AGGLUTINATION:
• Partial Hemolysis- Pink to red supernatant. With cell
button 1. Sensitization: initial binding of antigen and
- Not all of RBCs are hemolyzed. antibody; deposition of complement fragments
into the surface of red blood cell
• No Hemolysis- Clear supernatant with cell button. No
touched of red or pink. ➢ When the antibody binds the RBC
antigen, that is sensitization.
➢ When interpretating blood banking test, check ➢ When you only have sensitization, can
for agglutination and hemolysis. Dislodge tube you see visible clumping already? No.
when checking agglutination but in hemolysis ➢ This is either the antibody sensitizing
bawal galawin ang tube because RBCs will the RBC or a complement sensitizing
mix in supernatant, so mawawala yung the RBC
supernatant. 2. Lattice formation: formation of networks of
➢ Unang titignan ang hemolysis kesa sa antigen-antibody complexes (immune
agglutination. Bago mag-interpret kukunin ang complexes)
test tube and bawal i-shake para hindi humalo ➢ The antibody that sensitized RBC must
RBCs sa supernatant and hindi mamula; tignan form a lattice or a network and these are
agad ang supernatant. Kapag hindi pa the one that will be seen as visble
nagagalaw ang cell button at pink or red ang agglutination or visible clumping.
color meaning may hemolysis.
➢ Ang grading ng hemolysis ay CH (complete FACTORS AFFECTING SENSITIZATION:
hemolysis) or PH (partial hemolysis) 1. Serum to cell ratio: ratio of antibodies to antigens in
➢ Kapag walang hemolysis, check for the red cell
➢ Ideal serum to cell ratio in blood banking tests
agglutination.
should be 40:1 to favor sensitization
➢ Dislodge the cell button then check if there’s a
clump (small, medium large). Kapag walang • Mix 2 gtts of antibody and 1 gtts for antigen
nakitang clump with naked eye>> check under • In blood typing- 2 gtts of anti-sera and 1 gtts of
microscope. Report it as 0 kapag walang nag- patient’s RBC
clump kahit isa. • In major crossmatching- 2gtts of patient’s
serum and 1 gtts of Donor’s RBC
ANTIGEN-ANTIBODY REACTION • The higher the serum to cell ratio (ratio of AB to
AG) the higher the chance to sensitized RBC and
All antigen-antibody are governed by the Law of MASS collide AB
ACTION which states that Ag-AB reactions are reversible • Sometimes 133:1 ang ginagamit (3gtts of AB and
2 gtts of AG) Pero routinely, 40:1 ang ginagamit.
• AFFINITY: initial force of attraction between a single ➢ Antibodies==plasma/serum/anti-sera
binding site on the antibody and the antigen. Not that ➢ Antigens== RBC surface
strong compare with avidity
• AVIDITY: Sum total of all the forces of attraction NOTE: Increasing the concentration of antibody will increase
between a multivalent antigen and multivalent antigen
and multivalent antibody. (Stronger force) the probability of collision events with the corresponding red
• SPECIFICITY: antibodies will only react with antigens cell antigen
that have caused their production
2. Incubation time- incubate first reaction before
IMPORTANT NOTE: Antigen-Antibody reactions are interpreting results
specific. ➢ Incubation gives time for the Antibody to
They follow concepts of a lock and key. For example, Anti-A sensitized the corresponding RBC antigen;
antibody will bind A-antigen. Anti-B antibody will not react with kaya there must be an appropriate incubation
A-antigen time So, follow the SOP (kapag sinabing 15
minutes sundin ‘yun)
Intermolecular Forces that bind AG and AB together
1. Ionic Bonds- between positively charged and
3. Incubation temperature
negatively charged particles
➢ Will depend on the class of immunoglobulin;
Antigen== negatively charged kapag IGM>> room temperature and IGG>>
Antibody== positively charged warm (37’ Celsius)
2. Hydrogen bonds- occurs between 2 polar ➢ Blood banking test always done in 3 phases.
substances First the room temperature phase (IGM).
3. Hydrophobic bonds- occurs between 2 non-polar Second, incubation at 37’ Celsius (IGG). Lastly,
substances AHG phase.

B. LEGASTE & M. VALENCIA – TRANSCRIBERS

 4. pH: ideal is 6.7-7.2 (Average of 7.0) That is why the most commonly used sample in
BLOOD Bank test is 2-5% RCS (RED CELL
5. Ionic strength of the medium- decrease ionic SUSPENSION)
strength of medium to favor AB uptake by RBCs ➢ TOMATO RED OR CHERRY RED
➢ Use LISS (low ionic strength saline)

NOTE: decreasing the ionic strength of the medium will favor % Red Cell Suspension=
antibody uptake by RBCs
amount of washed RBC x 100
FACTORS AFFECTING LATTICE FORMATION:
Total Volume
1. Zeta Potential: Force of repulsion among RBCs
when placed in NSS or physiologic saline Total Volume= amount of washed RBC + amount of
NSS
➢ Under normal circumstances, RBCs has
negative charge on their surface because of the
sialic acid.
➢ Opposites charges attract and same charges ➢ Kapag pure blood, it contains millions of RBCs
repel. for a drop of blood. It means that kapag pure
➢ All of the RBCs are negative charge and blood, it outnumbers the amount of Antibody in
because of that they will repel each other (they the anti-sera (post zone) that will lead false
were far from each other). negative result.
➢ Example, theses RBCs are already sensitized ➢ Bakit of all concentrations 2-5% ang ginagamit?
by antibody; because they are far from each • Gumagamit ng commercially available anti-sera
other it is harder to form lattice. So, to promote in blood bank that contains known antibody.
lattice formation. zeta potential must be Example anti-A antisera na may lamang anti- A
decrease antibody. ‘Yung lamang antibody sa bawat anti-
Enahancement/ Reaction Media or Potentiators sera na ginagamit sa blood bank ay standardized
ang amount. And every person, hindi pare-
a) 22% Bovine Serum Albumin: Neutralize the pareho ang RBC count. So kaya 2-5% ‘yung
negative charge on the RBC (30 minutes) number ng RCS is because the number of RBCs
➢ Kapag wala nang negative charge, RBCs are in 2-5% RCS ay kapareho sa bilang ng
now close to each other which will make them antibodies sa mga commercially available na
easier to form lattice formation. anti-sera na ginagamit sa blood bank.
b) Low ionic strength Saline (LISS): Decreasing ➢ For example, may 100,000 na lamang antibody
the incubation time (10-15 minutes) sa anti-A antisera; yung laman na RBC sa 2-5%
c) Polyethylene glycol (PEG): Removes water to RBC suspension are more or less the same,
concentrate the antibody. 100,000 din. Meaning, the red cell suspension
d) Enzymes (papain, ficin, bromelin): enhance or ensures that the test that are being done is in
destroy certain red cell antigens. the equivalent zone.
Recap:
➢ Blood typing slide method in first year, pure blood ang
2. Optimum concentration of antibody and antigen
ginamit straight from skin puncture which gives us a
NOTE: Lattice formation only occurs at EQUIVALENCE disadvantage at pwedeng maging false negative dahil
zone pwedeng nasa postzone.
➢ In sensitization, the ratio of antibody is higher ➢ For example, nasa postzone reaction ka, kaya ‘yung
than the ratio of antigen while in lattice anti-b negative and anti-A negative, ang interpretation
formation the amount of antibody is equal to mo ay type O dahil walang reaction na na-occur.
antigen. ➢ Before interpretating results of blood type, centrifuge
➢ Doing the blood bank test at PROZONE first the test tube and gel card that will make RBC
(excess antibodies) and POSTZONE (excess closer to each other and promote lattice formation.
antigens) will lead to false negative results
➢ If there are excess antibody and excess
REFERENCES:
antigens, there’s no lattice formation== no
• Notes from Sir Joseph Joy Banzon, RMT, MPH
agglutination. In other words, PROZONE and
POSTZONE has no agglutination.

B. LEGASTE & M. VALENCIA – TRANSCRIBERS