IMMUNOHEMATOLOGY (BB) LEC

TOPIC 1: REVIEW OF IMMUNOSEROLOGY CONCEPTS AND

APPLICATION TO IMMUNOHEMATOLOGY

ANTIGEN VS. IMMUNOGEN

ANTIGEN - any substance which when introduced to the

host which can bind to a specific antibody

IMMUNOGEN - any substance that can trigger an immune

response

Immune response may entail activation of the cellular

components of immune system as well as the production
of specific antibodies.

TWO PROPERTIES OF AN ANTIGEN:

1. Specific reactivity
2. Immunogenicity

HAPTEN – incomplete antigen. Can react with specific

antibody; not immunogenic. May become immunogenic
when bound to ________________ CHARACTERISTICS OF BLOOD GROUP ANTIGENS:
1. Foreign to the host
EPITOPE – the particular area in an antigen • Introduced by: blood transfusion, tissue
molecule that confers specificity to it; it is also referred to transplantation, infection
as immunodominant molecule. The antibody response is
directed against this molecule. The specific antibody 2. Sufficient molecular size
recognizes this, and reacts to it. • Antigens on RBC - RBC as carrier of the
antigens
BLOOD GROUP ANTIGENS
3. Chemical complexity
• Proteins, glycoproteins are highly
BLOOD GROUP ANTIGENS
immunogenic
➢ Widely distributed mainly on the red cells, o Protein antigens are the most
throughout the tissues cells, white cells and immunogenic in nature
platelets, and in body fluids.
o they are not only found in blood 4. Structural stability
➢ ABH antigens in tissues makes them important to • Not easily degraded; red cells must not be
be considered in organ transplants lysed during testing
➢ Some blood group antigens are only adsorbed on
the red cells; they are secreted by epithelial cells BLOOD GROUP ANTIBODIES
into the body fluids (e.g. ABH, Lewis)
➢ Protein, large polysaccharide, glycoproteins or
GAMMA GLOBULINS
glycolipids
➢ Represent the blood group phenotype ➢ Produced in response to antigenic stimulation.
o H antigen is the precursor to ABO blood ➢ How?
type • Exposure through pregnancy
o Bombay phenotype – individuals who • Exposure through transfusion
does not have H antigen • Exposure through transplantation
o Integral protein – found in plasma ➢ Essentially of IgM and IgG classes; some IgA
membrane ➢ Characterized with specific reaction with blood
o An absence of some blood group group antigenic determinants / epitopes/
antigen may be translated to different immunodominant molecules
abnormalities
o Rh antigens serve as transporters → IgM antibodies important in blood banking include:
transport of CHO inside and outside • anti-A, anti-B, anti-H, anti-I, anti-i, anti-M, anti-N,
➢ Immunogenic, antigenic anti-Lewis, anti-Lutheran, and anti-P.
➢ Determined by blood group genes
➢ Rh antigens are integral structural component of IgG antibodies important in blood banking include:
the RBC membrane; if these antigens are missing, • anti- Rh(D), anti-S, anti-s, anti-Kell, anti- Duffy, and
the membrane becomes defective and results in anti-Kidd
hemolytic anemia.
IgA antibodies
• anti- Le, anti-A; anti-B

NOMENCLATURE OF ANTIBODIES
➢ Antibodies in immunohematology- use the prefix
anti… then - (dash), followed by the specificity.
• Examples: anti- A; anti-D; anti-Kell; anti-Kidd
o IgM (largest)
o IgG (transport to placenta)
➢ The specificity is with a capital letter if it named
after a person;

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IMMUNOHEMATOLOGY (BB) LEC
➢ In Rh-Hr nomenclature, the letters may be • Placental transfer (IgG)
capitalized or small letters. Examples: anti-D, anti- • Bind on receptor sites on tissues and other
C, anti-c, anti-E, anti-e cells

IMMUNE RESPONSE

IMMUNE SYSTEM
➢ activated by the presence of foreign antigen, or
abnormal autoantigen.
➢ Cells involved:
• APCs (antigen presenting cells) =
macrophages, dendritic cells, monocytes
• Lymphocytes = T helper, T cytotoxic, B
• Natural killer, neutrophils, eosinophils,
basophils, platelets
TYPES OF ANTIBODIES:
TYPES OF IMMUNE RESPONSES:
NATURALLY OCCURRING ANTIBODY – produced in 1. Direct B cell stimulation IgM antibodies
response to antigen stimulation from the environment 2. APC and T helper cell mediation
whose specificity is unknown / not definite (bacteria, • Humoral response: IgM and IgG antibodies
pollen, fungi, etc.); non-red cell stimulated; usually IgM • Cellular response: cytotoxicity by T cells, NK
cells
IMMUNE ANTIBODIES – produced from red cell antigen 3. Complement activation (by Ab-Ag complex, or
stimulation; IgG directly by pathogens, other substances)
o Hemolytic Disease of Fetus and Newborn
ISOAGGLUTININS – antibodies against red cell antigens, (HDFN) → blood incombatility
where these are differently distributed among the 4. Inflammatory response
members of the same species; cause specific clumping of • Ab-mediated
cells. Ex: Anti-A, Anti-B • Inflammatory cell stimulation

CLINICALLY SIGNIFICANT ANTIBODY - associated with HTR, BLOOD GROUP/TISSUE ANTIGEN STIMULATION
HDN and AIHA.
• PREGNANCY – incompatible blood type of fetus
IRREGULAR – Ab not expected to be found in serum of to that of mother (baby’s antigen foreign to the
patient; present in a very small human population only mother) E.g. Baby is D(+), mother is D(-)
• TRANSFUSION- incompatible blood type between
AUTOANTIBODIES – abs that react to red cells of the host, patient (recipient) and donor blood
as well as to red cells of other individuals; produced • TISSUE/ ORGAN TRANSPLANTATION –
through stimulation by bacterial or viral infections. incompatible type between patient (recipient)
and donor tissue
PASSIVELY ACQUIRED ANTIBODY - Antibodies that are • BACTERIAL / VIRAL INFECTION – autoantibodies
acquired through transfusion are produced, that detect red cell antigens
of plasma

HEMOLYSIS -Abs that activates the complement, and

cause red cells to lyse.

________________– ab that activates the complement

when it forms a complex with specific red cell antigen; IgM
in nature; hemolysis is produced when specific red cells are
reacted upon

________________– does not activate the complement

system;
IMMUNE RESPONSES RELEVANT TO IH:
COMPLETE ANTIBODY– ab that coat the red cells, and 1. Antibody production IgM and IgG
eventually lyses them; IgM 2. Hemolytic reaction mediated by Ab-Ag and
complement (cascade pathway)
______________– ab that only coat the red cell; but not 3. Inflammatory reaction tissue necrosis (organ
capable to lyse it. transplantation)
4. Allergic reaction mediated by IgE with basophils
WARM ANTIBODY - ab that best reacts at 37 C. due to plasma proteins received in transfusion

COLD ANTIBODY -ab that best react at 4C to ambient room ANTIGEN-ANTIBODY REACTION
temperature., (below 30C) MANIFESTATIONS OF ANTIGEN – ANTIBODY REACTION

ANTIBODY 1. Red cell agglutination

➢ Primary function: to bind with antigen • Clumping of red cells
➢ Secondary functions/ biological effector • Various grades of strength of reaction
functions: • (4+, 3+, 2+, 1+, +/-)
• Complement fixation/ activation → hemolysis
of red cells 2. Hemolysis (red cell destruction- Hb)

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• Red tinge of supernatant
• Minimal red cell button or none at all
• May be misread as negative

3. Mixed field agglutination

• Few red cells are agglutinated, amidst a
general field of un-agglutinated or free cells.
• Appear as 2+, mf.
Indications:
• Weak antigens in subgroups, example:
A3
• Acquired B phenomenon, example: o ABO Blood Typing gold standard is using TUBE
colon cancer METHOD
• Chimeras
o Tubes must be new because detergents or
• Mixed blood in transfusion
residues may result to erroneous results
o GEL CARDS → very critical in blood banking

NON- SPECIFIC AGGREGATION OF RED CELLS; NOT

(+) RESULT

ROULEAUX FORMATION
➢ Stacked-coin formation
o Product of high presence of proteins
present in plasma which affect the
zeta potential of red blood cells
Changes causing them to oppose and
maintaining proximity

➢ Found in patients with:

• Multiple Myeloma,
• Waldenstrom’s
• Macroglobulinemia, and
• Hyperviscosity Syndrome
➢ The red cells clusters and resembles a
macroscopic agglutination)
o Drop of NSS → this is to differentiate
Rouleaux from agglutination without
using microscope

NATURE OF AG-AB REACTIONS

LOCK AND KEY CONCEPT

➢ Interacting antigen and antibody-the antigenic
determinant fit in a cleft /space formed by the
combining site of the antibody (at the Fab region,
formed by variable regions of light and heavy
chains)

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IMMUNOHEMATOLOGY (BB) LEC
NON-COVALENT BONDS • Bring about increased rate of antibody uptake for
➢ Hold the Ag in the antibody combining site the Ag during sensitization shortens incubation
➢ Include hydrogen bonds, electrostatic bonds, Van period of 5 to 15 minutes.
der Waals forces and hydrophobic bonds
➢ Multiple bonding ensures the Ag bound to the Ab Time of Incubation
is tight and stable • To allow antigen/antibody reactions to reach
equilibrium
STAGES OF RED CELL AGGLUTINATION • 15 to 60 minutes

PRIMARY STAGE (ANTIBODY SENSITIZATION) Effect Of Antigen-antibody Ratio

• Amounts of antigen and antibody should be in
➢ Involves binding of the paratope/combining site
optimal proportions
of the Ab and the epitope/ determinant of the Ag
• Optimum ratio is 80 parts antibody to 1 part
(in a reversible reaction).
antigen.
• Excess in either antigen or antibody results to non
occurrence of lattice formation leading to a false-
negative result.

SECONDARY STAGE (LATTICE FORMATION)

➢ Multiple erythrocytes with bound antibodies form
a latticework through Ag-Ab bridges formed
between adjacent erythrocytes
➢ The lattice formed is the basis of all visible
agglutination reactions.

FACTORS AFFECTING THE SECONDARY STAGE OF

REACTION:

Zeta potential
• The surface of red cells carry a negative charge
due to the ionization of the carboxyl group of
NeuNac (N-acetyl neuraminic acid), also called
FACTORS AFFECTING THE PRIMARY STAGE OF REACTION: NANA or sialic acid
• In saline, red cells will attract positively charged
Effect of Temperature Na+, and an ionic cloud will form around each
• The nature of the bonds determines if the reaction cell. Thus, the cells repel each other and stay a
occurs better at colder or warmer temperature. certain distance apart.
• With hydrogen bonds- they are exothermic, • IgG antibodies cannot cause agglutination when
reaction occurs better at colder temperature zeta potential exists.
• Associated with carbohydrates antigen e.g. ABH, • To overcome zeta potential techniques are
Lewis, P1, I needed to
• If with hydrophobic bonding, the reaction is • neutralize these charges.
optimally reactive at body temperature, 37 C
• This type of bond is normally associate with protein
antigen
• e.g. Rh, Kell, Duffy

Effect of pH
• Optimum pH for most antigen-antibody reactions
→ 6.5 to 7.5
• Some antigens show stronger reactivity at lower
pH
• e.g. anti-M, stronger reaction below pH 6.5

Ionic Strength Effect of Centrifugation

• refers to charge concentration of the suspending • The antibody - sensitized red cells are subjected to
(reaction) medium. high gravitational force overcoming the natural
• influences the rate of formation of the antigen- repulsive effect of the red cells to one another
antibody complex • Closer physical proximity increases antigen-
• decreasing the ionic strength of the suspending antibody bridging
medium reduces the interfering effects of the
electrostatic barrier, thus better attraction Effect of Immunoglobulin Type
between antigen and antibody. • Most efficient for agglutination reactions.
• The physical size of IgM
Low Ionic Strength Saline (LISS) • The number of antigen binding sites of IgM
• Consists of 0.2 % NaCl molecules (valence 10) compared with IgG
(valence 2), increases the chances of random

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antibody-antigen collisions, leading to a greater
chance of effective red cell cross-linking.

Anti-human globulin
• ANTIHUMAN GLOBULIN REAGENT
• Produces a “bridging effect” by cross linking the
antibody-sensitized red cells

ENHANCEMENT MEDIA FOR Ag- Ab REACTIONS

A. ALBUMIN
The mechanisms:
1. Reduce the charge density (dielectric constant)
of the red cell suspending medium.
2. Reduce net repulsive force between red cells
3. It replaces the water hydration surrounding the
red cell

➢ IgG antibody able to span the gap between

individual positive red cells to produce
agglutination.

B. ENZYMES
The proteolytic enzymes:
1. papain (papaya)
2. ficin (figs)
3. bromelin (pineapples)
4. trypsin (lining of a hog's stomach)

➢ Release of sialic acid from the RBC membrane,

with subsequent decrease of negative charges
and zeta potential of RBCs.
➢ Order of effectiveness in detecting IgG
antibodies: ficin > papain > bromelin > trypsin

POLYETHYLENE GLYCOL
➢ Water- soluble polymer used with AHG to bring
sensitized cells close together and facilitate cross-
linking and enhancement of agglutination
reaction.
➢ Does not produce non-specific reactions.

POLYBRENE (hexadimethrine bromide)

➢ Positively charged macromolecule which cause
non-specific aggregation of sensitized red cells
with IgG antibody after incubation with LISS.

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IMMUNOHEMATOLOGY (BB) LEC
THE RED CELL MEMBRANE PROTEINS
➢ Form a meshwork that traverse the lipid bilayer,
WHY DO WE STUDY THIS TOPIC?
called membrane skeleton
• Mainly, blood groups antigens are located in/on
➢ Spectrin, glycophorin and band 3 account for
the red cell membranes. The structure of the red
>60% of total membrane protein
cell membranes has certain roles for its
functionality. There are important things to
2 TYPES OF PROTEINS:
remember about the characteristics of red cell
a. “Integral” membrane proteins- extend from the
membranes.
outer surface and span the entire membrane to
• The main purpose of blood transfusion is to supply
the inner cytoplasmic side of the RBC
red cells which should remain viable and
o May act as channels/
functional in the transfused patient/recipient.
transporters
• Therefore, we have to understand the nature of
b. “Peripheral” proteins – located and limited to the
RBC membranes.
cytoplasmic surface of the membrane forming
the RBC cytoskeleton
RED BLOOD CELL o Form the RBC cytoskeleton
➢ MAIN FUNCTION OF RED CELLS: o Cytoskeleton → very capsule of
To transport oxygen to tissues, and bind cells, prevents accidental
carbon dioxide from tissues to be brought to bursting of cells
the lungs for excretion.
➢ CRUCIAL FOR RBC SURVIVAL AND FUNCTION:
1. Normal chemical composition and structure
of the RBC membrane
2. Hemoglobin structure and function
3. RBC metabolism

RED BLOOD CELL MEMBRANE STRUCTURE

ERYTHROCYTE MEMBRANE
➢ RBC membrane is a semipermeable lipid bilayer
supported by a protein mesh-like cytoskeleton
structure.
o Phospholipid head (hydrophilic)
o Fatty acid (hydrophobic)
o RBC is semipermeable
o Fatty acids → prevents the passage of
water- and water-soluble substances in
order to maintain isotonicity and in order
to prevent hemolysis

LIPIDS
➢ phospholipids= 50 %; cholesterol= 40 %;
glycolipids= 10 %
o 40% of RBC

PHOSPHOLIPIDS FORM A BILAYER

➢ hydrophobic (nonpolar) tails towards inside of cell
➢ hydrophilic (polar) head groups towards outside
(extracellular) or the inside (cytoplasmic) surface
BEHAVES AS SEMISOLID, WITH ELASTIC AND VISCOUS ➢ the outer leaflet (external layer) contains
PROPERTIES electrically neutral glycolipids (sphingomyelin)
➢ Important and critical for survival in the circulation and choline phospholipids (phosphatidyl choline);
for 120 days going through numerous cycles and while
passages through narrow veins and sinusoids in ➢ the inner leaflet (internal cytoplasmic layer)
the spleen. predominantly contains phosphatidyl serine
o In order for the RBC to fit in sinusoid which is negatively charged.
(capillaries) by stretching o Phosphatidyl serine → cell death marker,
➢ Critical in RBC deformability and permeability cell is >120 days, phagocytose
(crucial / critical characteristics) o SCRAMBLASE ENZYMES → asymmetric
(random) distribution
BIOCHEMICAL STRUCTURE o FLIPPASE → transfer phosphatidyl serine to
outer to inner
• The cell membrane consists of: protein, lipids, and o FLOPPASE → able to exchange
carbohydrates phosphatidyl serine for your neutral
• Ratio of: 52% (Proteins) phospholipid
40% (Lipids) o FLIPPASE & FLOPPASE →
8% (Carbohydrates) - Action: mainly drive by ATP
- ATP: acts as energy for RBC
- When RBC reached its days, ATP
diminishes as well as the function →

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phosphatidyl serine will go to outer PERMEABILITY
leaflet ➢ Important to prevent colloidal hemolysis and
- Death marker – phagocytose control the volume of RBC: favorable to active
- Main driver for RBC deformability cation transport
CARBOHYDRATES ➢ Permeable to water and anions, like Chlorides
➢ Forms the glycocalyx, serving as a negatively and bicarbonate
charged barrier (10 A thick), around the outside of ➢ Relatively impermeable to cations like Na and K
the RBC membrane ➢ Intracellular: extracellular ratio of Na -1:12; K -25:1
o Glycocalyx → prevents the RBC from ➢ Cationic pump for active transport (requiring ATP)
sticking to each other and it has a zeta of Na and K
potential ➢ Ca is transported outward from cell, depends on
➢ Important in preventing cells from adhering to one Ca- ATPase pump
another, or adhering to the endothelium ➢ Calmodulin (calcium-binding protein)- control the
ATPase pump; prevent excessive intracellular
BLOOD GROUP ANTIGENS ON THE MEMBRANE buildup which leads to rigidity.
➢ When ATP is depleted, Na and Ca accumulate
➢ Majority carried on transmembrane proteins intracellularly, K and water get out of the cell →
➢ Few antigens carried on glycosylphosphatidyl- dehydration and rigidity of cell → easily
inositol (GPI)- linked proteins or on glycolipids sequestered by the spleen, thus decrease in RBC
➢ Some transmembrane proteins interact with other survival.
transmembrane blood group antigens
➢ Ex:(band 3 and GPA) like Kell and Kx, Rh and METABOLIC PATHWAYS
RhAG
➢ Some antigens are adsorbed from plasma: Lewis, ANAEROBIC METABOLIC PATHWAY IS UTILIZED
Chido ➢ Anaerobic because oxygen is supposed to be
delivered to the tissues, therefore should not be
consumed
➢ Red cell does not have mitochondria and nucleus
to generate the energy for its metabolism
➢ Mainly derived from glucose → anaerobic
glycolytic pathway

EMP PATHWAY
EMBDEN MEYERHOF PARNAS PATHWAY
➢ Glucose → lactate
➢ 2 ATP generated; 90% ATP needed by RBC
o 90% ATP needed by RBC

PENTOSE PHOSPHATE PATHWAY/SHUNT

➢ The pentose phosphate pathway (also called the
phosphogluconate pathway and the hexose
monophosphate shunt); Warburg-Dicken’s
Pathway)
o Shunt – bypass
o Able to prevent oxidative stress
➢ This metabolic pathway run parallel to glycolysis.
OTHER PROPERTIES OF THE RED BLOOD CELL MEMBRANE ➢ It involves the oxidation of Glucose-6-Phosphate
to 6-Phosphogluconic acid which in turn is
converted into pentose phosphates. In this
DEFORMABILITY
pathway glucose-6-phosphate is directly oxidized
➢ Red cell must be easily deformed/change in without entering glycolysis, hence it is also known
shape → advantageous when these pass-through as Direct Oxidation Pathway or Hexose
small openings or channels, as in the spleen Monophosphate Shunt.
o SPLEEN → graveyard of RBC ➢ It generates NADPH and pentoses (5- carbon
sugars) as well as ribose 5-phosphate, a precursor
CAUSES OF LOSS OF DEFORMABILITY/RIGIDITY OF CELL: for the synthesis of nucleotides (RNA and DNA)
• Loss of ATP→ decreased phosphorylation of ➢ When the PPP is deficient functionally, the amount
spectrin of reduced glutathione becomes insufficient to
• Increase deposition of calcium onto cells neutralize intracellular oxidants.
• Examples: spherocytes, “bite cells” cell
become easily lysed

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➢ This results in the denaturation and precipitation of
globin as aggregates called HEINZ bodies within
the cell.
➢ Heinz bodies within the RBC → deformability is lost
→ easily lysed in the spleen or cause blockage in
the capillaries.

METHEMOGLOBIN REDUCTASE PATHWAY

➢ It serves to maintain the iron (in the heme of
hemoglobin) in the ferrous state (Fe2+) to be more
functional.
➢ In the absence of the enzyme (methemoglobin
reductase and coenzyme NAD (nicotinamide
adenine dinucleotide), there is the accumulation
of methemoglobin resulting from the production
of more ferric iron (Fe3+).
➢ Metheme can NOT bind with oxygen
o Fe (3+) → Fe (2+)

LUEBERING-RAPAPORT PATHWAY
➢ Allow the production of 2,3-diphosphoglycerate
(2,3-DPG) within the red cells.
➢ Controls the release of oxygen from hemoglobin
to the tissues.
➢ Increased 2,3 DPG → easier release of O2 (Right
shift)
➢ Decreased 2,3 DPG → stronger affinity to the Hb
→ less efficient release into the tissue (Left shift)

RAIZZA ELOISSE F. CARVAJAL | BMLS III-1