MEDICAL TECHNOLOGY ASSESSMENT PROGRAM 2

IMMUNOHEMATOLOGY
LECTURE 1: BASIC CONCEPTS OF IMMUNOHEMATOLOGY

Menissa S. Acierto, RMT, MEd

BLOOD
Components:
Fluid that provides the major transport system in the body
Composed of solid (cellular components) and liquid (plasma or serum component
Liquid portion
Plasma – liquid portion of blood sample collected with an anticoagulant while
serum is the liquid portion of a blood sample that has clotted
Plasma makes up approximately 55% of the blood volume and is composed of
greater than 90% water
Serum is the liquid component most often used in blood bank testing
Cellular components suspended in the plasm include erythrocytes, leukocytes and
thrombocytes
BLOOD
Functions:
Main function is the transport of :
Oxygen to the tissues and carbon dioxide to the lungs for expiration
(this is the function of the erythrocytes)
Nutrients, hormones, and chemical substances to the tissues
Waste products to site of removal
Final important function of blood is coagulation; coagulation protects
the body by preventing bleeding
White cells are involved in phagocytosis and immunity
BLOOD
Blood circulation:
Blood flows through the body in 2 distinct circulations: pulmonary and
systemic, the heart is the pump for each circulation
Pulmonary circulation
Oxygen and carbon dioxide exchange between the blood and the
inspired air occurs in the pulmonary circulation
Systemic circulation
Oxygenated blood is pumped by the heart throughout the body
providing the oxygen required for the various metabolic processes of
tissues
BLOOD
Hemoglobin:
Oxygen enters the red cells in the lungs and binds to an intracellular
protein called hemoglobin
Hemoglobin consists of a porphyrin ring with a central iron atom (heme)
and a globular protein made up of 2 alpha and 2 beta globin chain (four-
sub units) each of which carries one hem group
Each red cell contains many hemoglobin molecules
BLOOD
Hemoglobin:
BLOOD
Oxygen-dissociated curve (normal sigmoid curve):
The relationship between oxygen concentration in the blood (pO2) and
the percentage of oxygen bound to hemoglobin (sO2) form a sigmoid
curve
A raise in pH increases the hemoglobin/oxygen affinity making it more
difficult for hemoglobin to release oxygen; consequently, hemoglobin
holds on to the oxygen molecule maintaining a higher saturation; this
results in a shift of the curve to the left
BLOOD
Oxygen-dissociated curve (normal sigmoid curve):
Red cells produce 2,3 diphosphoglycerate (2,3 DPG); this substance
diminishes the hemoglobin/oxygen affinity and facilitates the release of
oxygen from the red cells; therefore the oxygen dissociation is shifted to
the right
The level of 2,3 DPG progressively decreases as blood donor blood is
stored; theoretically, this decrease can result in reduced oxygen delivery
to the tissues and a shift of curve to the left; most transfused patients
are unaffected by this change as the 2,3 DPG in donor red cells returns to
normal within 24 hours of infusion
BLOOD
BLOOD
Red blood cell membrane:
The RBC membrane is a semi-permeable lipid bi-layer (mainly
phospholipids) supported by a protein meshlike cytoskeleton structure
The RBC membrane consists of 2 kinds of proteins, the integral proteins
(or transmembrane proteins) and peripheral proteins
Integral proteins extend from the outer surface and span the entire
membrane to the inner cytoplasmic side of the RBC: glycophorin A,
glycophorin B, glycophorin C, anion-exchange-channel protein
Peripheral proteins are located and limited to the cytoplasmic surface
of the membrane forming the red cell cytoskeleton: spectrin, actin
(band 5), Ankyrin (band 2.1), band 4.1 and 4.2, band 6, adducin
BLOOD
BLOOD
RBC characteristics:
Two important characteristics are deformability and permeability
Deformability of RBC
The loss of RBC flexibility and deformability is due to:
Decrease in the phosphorylation of spectrin (due to decrease ATP
levels)
Accumulation/increase deposition of membrane calcium
BLOOD
RBC characteristics:
The loss of deformability leads to:
Easy extra vascular sequestration and lysis of RBC at the sinusoidal
spaces of the spleen
Spherocytosis (spherocytes are formed due to the loss of RBC
membrane and consequently a reduced surface-to-volume ratio)
Formation of bite cells (due to the removal of a portion of RBC
membrane leaving a permanent indentation in the remaining cell
mebrane)
Survival of these forms is shortened.
BLOOD
RBC characteristics:
Permeability of RBC
The RBC membrane is freely permeable to water and anions; chloride and
bicarbonate can traverse the membrane in less than a second
The RBC membrane is relatively impermeable to cations such as sodium and
potassium; the RBC volume and water homeostasis are maintained by controlling
the intracellular components of sodium and potassium
The erythrocyte intra cellular-to-extra cellular ratios for sodium and potassium are
1:12 and 25:1 respectively
When RBC are depleted, calcium and sodium are allowed to accumulate
intracellularly and potassium and water are lost, they become dehydrated and
subsequently sequestered by the spleen, resulting in a decrease in RBC survival
BLOOD
RBC characteristics:
Metabolic pathways of RBC
The RBC metabolic pathways that produce ATP are mainly anaerobic because the
function of RBC is to deliver oxygen and not to consume it
Since mature RBC is non-nucleated and no mitochondrial apparatus for oxidative
metabolism, energy is generated almost exclusively by glycolysis (glucose
breakdown)
Metabolic pathways are divided into:
Anaerobic Glycolytic Pathway
Pentose Phosphate Pathway
Methemoglobin Reductase Pathway
Luebering Rapaport Shunt
BLOOD
RBC characteristics:
Metabolic pathways of RBC
Anaerobic Glycolytic Pathway
Generates about 90% of the ATP needed by the RBC
Pentose Phosphate Pathway
Increased following:
Increased oxidation of glutathione
Decreased activity of the anaerobic glycolytic pathway
Produces approximately 10% of the ATP needed by the RBC
When the pathway is deficient, the amount of reduced glutathione becomes
insufficient to neutralize intracellular oxidants
The result is denaturation and precipitation of globin as aggregates (Heinz
bodies) within the cell; Heinz bodies makes RBC less deformable than normal
RBC
BLOOD
RBC characteristics:
Metabolic pathways of RBC
Methemoglobin Reductase Pathway
This pathway is necessary to maintain the heme iron to hemoglobin in the
ferrous functional state
In the absence of the enzyme methemoglobin reductase and the action of NAD,
there is accumulation of methemoglobin, which results from a conversion of
ferrous to the ferric form
Methemoglobin is a non-functional form of hemoglobin and a loss of oxygen
transport capabilities
BLOOD
RBC characteristics:
Metabolic pathways of RBC
Luebering Rapaport Shunt
This permits the accumulation of 2,3 diphosphoglycerate (2,3 DPG)
The large amount of 2,3 DPG found within RBC has a significant effect on the
affinity of hemoglobin for oxygen
BLOOD
RBC preservation:
Successful transfusion
75% of cells that have been transfused should remain viable for 24 hours for
transfusion to be considered successful
Storage of blood
Storage of blood may lead to various biochemical changes
Decrease in pH
Building of lactic acid
Decrease in glucose consumption
Decrease in ATP levels
Loss of red cell function
BLOOD
RBC preservation:
2,3 DPG levels
As blood is stored, 2,3 DPG levels decrease
There is a shift to the left of the hemoglobin dissociation curve, and less oxygen is
delivered to the tissues
Approved preservative solutions (added to whole blood)
Acid-citrate dextrose (ACD), CPD, CP2D, are approved preservative solutions for
blood storage at 1-6oC for 21 days
Since ACD has a lower pH, 2,3 DPG is lost early during storage
CPD is more superior than ACD in preserving 2,3 DPG
BLOOD
RBC preservation:
Approved preservative solutions (added to whole blood)
Adenine is incorporated to CPD (forming CPDA-1) in order to increase ADP levels,
thereby driving glycolysis toward the synthesis of ATP
Storage time
ACD and CPD – 21 days
CPDA-1 – 35 days
CPDA-2 and SAG-M – 42 days
BLOOD
Composition of Approved Preservatives
ACD CPD CPDA-1
Trisodium citrate (g) 22.0 26.30 26.35
Citric acid (g) 8.0 3.27 3.27
Dextrose (g) 24.5 25.50 31.90
Monobasic sodium phosphate (g) - 2.22 2.22
Adenine (g) - - 0.27
Water (ml) 1000 1000 1000
Volume/100 ml blood (ml) 15 14 14
Approximate volume of preservative pH bag (ml) 67.5 63 63
Initial pH sol. 5.0 5.6 5.6
pH of blood on initial day drawn into storage bag 7.0 7.2 7.4
Storage time (days) at 1-6oC 21 21 35
BLOOD
RBC preservation:
additive solutions (added to packed red cells)
A new blood collection system employs a primary bag containing standard
anticoagulant and an accessory bag (or satellite bag) containing additive solution
After the plasma is removed from a unit of whole blood, the additive solution is
added to red cells to provide nutrients for improved viability
In general the additive solutions employed are composed of: Saline (S), Dextrose or
Glucose (G), Adenine (A)
Hogman (Sweden) and Lovric (Australian) additive solutions differ only in that Hogman
uses standard CPD anticoagulant in the primary bag with an additive solution containing
the SAG
Hogman was modified with the addition of mannitol to maintain the integrity of RBC
membrane
BLOOD
RBC preservation:
additive solutions (added to packed red cells)
Lovric doubled the dextrose concentration with the additive solution containing
saline, adenine, glucose, tri-sodium citrate, citric acid and sodium phosphate
The additive solutions licensed in the US are:
Adsol (AS-1) Fenwal Laboratories
Nutrical (AS-3) Medsep Corporation
Optisol (AS-5) Terumo Corporation

ADSOL contains buffered adenine, glucose, mannitol (to retard hemolysis)

Storage time is 42 days
BLOOD
RBC preservation:
additive solutions (added to packed red cells)
Formulation of additive solutions
AS-1 AS-2 AS-5

Adenine (mM) 2.00 2.22 2.22

Glucose (mM) 11.00 55.51 45.41

Mannitol (mM) 41.20 - 28.82

NaCl (mM) 154.00 70.1 150.04

Na2HPO4 -- 23.00 --

Primary bag CPD CD2D CPD

anticoagulant
BLOOD
RBC preservation:
Rejuvenation solutions
Generally, red cells stored in the liquid state for less than 3 days after expiration
date can be rejuvenated by incubation for 1 to 4 hours at 37oC with rejuvenation
solution
Rejuvesol (Cytosol Laboratories) is the only FDA-approved rejuvenation solution;
consists of phosphate, inosine, pyruvate and adenine (PIPA)
BLOOD
RBC preservation:
Red Cell Freezing
Red cell freezing is primarily done for autologous units and storage of rare blood
types; individuals may donate blood for their own future use (autologous
transfusion)
Red cell freezing involves the addition of a cryoprotective agent to red cells that
are less than 6 days old
Glycerol is the most commonly used; it is added to red cells slowly with mixing to
enable glycerol to permeate the red cells; the red cells are rapidly frozen and
stored in a freezer with:
High concentration glycerol 40% w/v (this is most commonly done)
Low concentration glycerol 20% w/v
BLOOD
RBC preservation:
Red Cell Freezing

High glycerol Low glycerol

Initial freezing temperature -80oC -196oC

Type of freezer mechanical Liquid nitrogen

Maximum storage -65oC -120oC

temperature
Shipping requirement Dry ice Liquid nitrogen

other Can be thawed and refrozen critical

BLOOD
Blood preservation:
Blood substitutes are of 2 categories:
Hemoglobin-based oxygen carrier
Perfluorochemicals (PFC)
Hemoglobin-based oxygen carriers include stroma free hemoglobin solution (SFHS),
chemically modified hemoglobin solution recombinant hemoglobin and
encapsulated hemoglobin
Perfluorochemicals are chemically inert but excellent gas solvents; they carry O2 and
CO2 by dissolving much as 40 to 70% oxygen per unit volume
BLOOD REPLACEMENT FLUIDS
Intravenous fluids (IV-fluids):
Have a variety of uses:
Provide the normal maintenance fluid requirements of a patient in whom the oral
route is unavailable
To provide replacement fluids for abnormal losses incurred as a result of surgery,
trauma or other pathology
Correct electrolyte disturbance or hypoglycemia
Act as vehicle for the administration of certain drugs
Maybe maintenance fluids and replacement fluids
BLOOD REPLACEMENT FLUIDS
Maintenance fluids:
Used to replace the normal physiological losses that occur in a patient through skin,
lung, feces and urine
Since a considerable proportion of these losses is water, maintenance fluids are
mainly composed of water in the form of dextrose solution (electrolytes maybe
added)
All maintenance fluids are crystalloid solutions, eg:
5% dextrose solution
4% dextrose in sodium chloride (0.18%)
Volume of maintenance fluid required by the patient depends on
Pyrexia
High ambient temperature or humidity when losses will increase
BLOOD REPLACEMENT FLUIDS
Replacement fluids (or plasma substitutes):
used to replace abnormal losses of blood, plasma or other extra cellular fluids by increasing
the volume of the vascular component
Used principally in the:
Treatment of patients with established hypovolemia (e.g. hemorrhagic shock)
Maintenance of normovolemia in patients with ongoing fluid losses (surgical blood loss)
Examples:
Balanced slat solutions (a solution of NaCl with electrolyte composition resembling that of
extracellular fluid)
NSS (0.9% NaCl)
Ringer’s lactate
Hartmann’s solution
All colloid solutions
GENETICS
Phenotype frequencies are expressed as percentage or decimal
Phenotype frequencies are determined testing red cells from large random population of the
same race. The percentage of positive or negative reaction is determined with a given known
antiserum
All possible phenotype frequencies for a given system totals to 100% or 1.00.
Sample problem:
The frequency of Jk(a+) persons is 77%. Consequently, Jk(a-) persons is 23%. If the patient has other
antibodies such as anti-c, anti-K, and anti-Jka then the calculation is as follows:
C = 20%
k = 91%
Jk(a-)=23%
Then 0.2 x 0.91 x 0.23 = 0.04
This means that 4 compatible units is obtained from screening 100 units of blood.
LECTINS AND PROLECTINS
Lectins are specific antibodies derived from plants; prolectins are derived from snails

Specificity Source
Anti-A Dolichus biflorus
Anti-B Banderaea simplicifolia
LECTINS
Fomes fomentarius
Anti-H Ulex europaeus
Anti-A Helix pomatia
Helix aspersa
Cepaea nemoralis
PROLECTINS Anti-A1 Euphrada pariomphala
Bradybaena fructicum
Anti-B Salmo irideus
Anti-H Eel
RED CELL ANTIGEN-ANTIBODY INTERACTION
Detection
Red cell antigen-antibody interaction in the blood bank may be detected by a number of serological
techniques that involve hemolysis or hemagglutination
Hemolysis
Occurs if the entire complement sequence is activated following antigen-antibody interaction
Many antibodies activate complement, but as the sequence frequently stops at C3, in vitro-lysis of the
red cells rarely occurs
Hemagglutination
Most commonly used indicator of an antigen-antibody reaction
Sensitization is the process whereby antibody is attached to the antigen on the surface of the cell
Hemagglutination occurs when bound antibody links to adjacent red cells forming clumps
IgG does not result in agglutination since it is too small to span the distance between 2 red cells
IgM can easily cause agglutination
RED CELL ANTIGEN-ANTIBODY INTERACTION
The Zeta Potential Theory
Under normal conditions, red cells do not agglutinate because of zeta potential
The red cell surface has a negative charge due to sialic acid molecules on the membrane; when the red
cells are suspended in saline, the cations are attracted to the negative red cell surface, forming a
repelling cloud around the cell; this electric repulsion between cells is known as the zeta potential.
Reducing the zeta potential allows the cells to approach each other facilitating agglutination
Enzymes remove some of the negative charge on the red cell resulting in a smaller cloud of positive
ions; the density of the cloud is reduced therefore zeta potential is decreased
Albumin dissipates some of the positive charge on the red cell result; therefore, few cations surround
the red cell and the zeta potential is reduced
Decreasing the concentration of cations in the medium decreases the density of ions around the cell;
this increases the rate of sensitization (Low Ionic strength solution – LISS)
RED CELL AGGLUTINATION
The factors affecting red cell agglutination include:
Antibody length
Antibodies vary in length
IgM – 1000Ao (angstroms)
IgG – 250 Ao
Zeta potential (electric charge in red cell surface)
Red cell surfaces are negatively charged due to sialic acid residues; when red cells
are suspended in media, cations becomes attached in a cloud-like effect around the
cell
The edge of this is known as the slipping plane and the electric potential at this
point is termed the zeta potential
The zeta potential maybe reduced by suspending red cells in macromolecule media
(albumin, PVP); the cells become closer together, allowing red cell antibody-red cell
bridges to be made, and thus allowing agglutination
RED CELL AGGLUTINATION
The factors affecting red cell agglutination include:
Position and number of antigen sites
Some antigens on red cell surface are situated in a more accessible location to antibody
hence agglutination is likely to occur
Red cell antigens vary in the number of antigen sites
A1 – 0.81-1.17 x 106
A2 – 0.24 – 0.29 x 106
B = 0.75 x 106
D = 9.9 – 3.3 x 103
Kell = 3.5-6.1 x 103
RED CELL AGGLUTINATION
The factors affecting red cell agglutination include:
Length of incubation time
Most blood group antigens possess relatively high binding constants, so that incubation
period of up to one hour is optimal for the detection of the reaction
Reaction temperature
in general, IgM antibodies react best in cooler temperature between 4-25oC, whereas, IgG
antibodies react more strongly at 30-37oC
Antibodies that react in vitro only at temperature 37oC have little clinical significance
Antibodies that do not agglutinate red cells or activate complement above 30 oC do not
cause destruction of transfused cells
RED CELL AGGLUTINATION
The factors affecting red cell agglutination include:
pH
Anti-D antibodies react more strongly at pH 6.5 to 7 while anti-M antibodies react
strongly at pH 6.0-6.5
Concentration of antigen and antibody
Both antigens and antibodies must be present in optimal concentration to obtain
the best visible reaction
If the antibody is in excess, it will result in a situation known as prozone
phenomenon, while if the antigen is in excess, postzone phenomenon occurs
GRADING AGGLUTINATION REACTIONS
When agglutination has occurred, it is necessary to determine the strength of the reaction
Standard system of grading reactions
Grade Description
0 (Negative) No clumps or aggregates
+ (weak) Tiny clumps or aggregates barely visible macroscopically or to the naked eye
1+ (plus 1) A few small aggregates visible macroscopically, background supernatant
cloudy
2+ (plus 2) Medium size aggregates, clear background
3+ (plus 3) Several large aggregates, clear background
4+ (plus 4) One solid aggregate, clear supernatant
MEDICAL TECHNOLOGY ASSESSMENT PROGRAM 2

IMMUNOHEMATOLOGY
LECTURE 2: BLOOD GROUP SYSTEMS: AN INTRODUCTION

Menissa S. Acierto, RMT, MEd

BLOOD GROUP ANTIGENS
Definition:
Refer to the genetically encoded antigen system on the erythrocytes, leukocytes,
platelets and plasma
Only relatively small number of blood group antigens is considered significant but
more than 600 have been described
BLOOD GROUP ANTIGENS
Immunogenecity:
The majority of the blood group antigens are immunogens - they are able to elicit
antibody-mediated immune response when introduced as a foreign substance into a
responsive host
The characteristics of antigens that determine their imunogenecity include the:
Degree of foreignness
Molecular size and configuration
Temperature, pH and ionic environment
Antigenic complexicity (measured by the number of available epitopes)
BLOOD GROUP ANTIGENS
Immunogenecity:
Blood groups differ in immunogenicity:
The A, B and D (Rho) antigens are certainly the most immunogenic and thus all
blood transfused must be matched for these antigens between the blood donor
and the recipient
After the D antigen, K is the next most immunogenic, with Fya and other antigens
within the Rh system ( C, c, E, e)
Other common blood groups are much less immunogenic (Fyb, Jka, Jkb and s)
BLOOD GROUP ANTIGENS
Chemical characteristics
Red cell antigens are usually proteins, glycoproteins, lipoproteins or glycolipids are
embedded to or protruding from the RBC membrane (except Lewis antigen)
Incidence of blood group antigens
Low incidence antigens
These antigens are found in less than 1% of the population
Cw, V, Kpa, Jsa, Lua, Mo, Vw, Dia, Wra and Cob are examples
These are called private or family antigens
High incidence blood group
These antigens are found in more than 99% of the population
Kpb, k, Jsb, Lub, I, Ge, Tja, Vel, Yta, Dib, Coa, and U are examples
They are also called public antigens
BLOOD GROUP ANTIGENS
Inheritance of Blood Group Genes
Autosomal co-dominance is the most common pattern or inheritance of blood group
genes
The genes are inherited equally by males and females and they are always expressed
If one inherits the A and B genes, both A and B antigens are detected on the RBC
The RBC antigens that are not fully expressed at birth include A, B, I, P Lewis and
Lutheran
BLOOD GROUP IMMUNOGLOBULINS
Immunoglobulins are protein molecules that are produced in response to antigenic
stimulation and that demonstrate specific antibody activity.
The majority of clinically significant blood group antibodies fall into IgG and IgM
immunoglobulin classes with occasional IgA forms.
Blood group antibodies are usually classified as:
Alloantibody – it reacts with a foreign antigen not present on the patient’s own RBC.
Autoantibody – it reacts with an antigen on the patient’s own cells and with that
same antigen on the cells of other individuals
BLOOD GROUP IMMUNOGLOBULINS
Alloantibodies to erythrocyte antigens are either:
naturally occurring – the antigenic stimulus for their production is unknown.
Naturally occurring antibodies appear in the serum who lack the corresponding
antigen
immune antibodies – they are produced as a result of immunization to foreign
erythrocyte antigens either by:
exposure through transfusion of blood components
through pregnancy (usually at the time of delivery)
MAJOR BLOOD GROUP SYSTEMS
ABO System
The ABO system differs from other blood group system and that it is the only one in
which there are naturally occurring antibodies in the plasma/serum.
The ABO system is the most important in transfusion therapy. Naturally occurring
anti-A and anti-B are potent hemolytic antibodies. Life-threatening acute hemolytic
transfusion reactions may occur from an ABO-incompatible transfusion.
They are also found on tissue cells, platelets, lymphocytes and in a soluble form in
the body fluids and secretors.
Rh Blood Group System
Among the antigen in the Rh system, the Rho(D) antigen is the most potent.
The presence of D antigen denotes Rh-positive blood. 85% of the US Caucasian
populations are Rh-positive and 15% are Rh-negative.
OTHER BLOOD GROUP SYSTEMS
The other blood group systems are divided into:
Antigen Systems that produce cold-reacting antibodies. These antibodies react at
temperature less than 37oC. They are not considered clinically significant since they
are less likely to occur in hemolytic transfusion reactions (HTR).

Systems Major Antigens

Lewis Lea, Leb
P P1
I I, i
MNSs M, N, S, s
OTHER BLOOD GROUP SYSTEMS
The other blood group systems are divided into:
Antigen systems that produce warm-reacting antibodies. They react at 37oC and are
considered as clinically significant because they cause hemolytic disease of the
newborn (HDN) and HTR

Systems Major Antigens

Kell K, k
Kidd Jka, Jkb
Duffy Fya, Fyb
MEDICAL TECHNOLOGY ASSESSMENT PROGRAM 2

IMMUNOHEMATOLOGY
LECTURE 3: THE ABO BLOOD GROUP SYSTEM

Menissa S. Acierto, RMT, MEd

HISTORICAL PERSPECTIVE
ABO blood group system is the first human blood group system discovered and
remains the most important blood group in transfusion practice
In 1901, Landsteiner described the first 3 blood groups in the ABO system: Group A,
Group B, and Group O
In 1902, Landsteiner associates Strule and Von Descatello discovered the fourth blood
group in the ABO system: Group AB
LANDSTEINER LAW
In 1900, Landsteiner discovered A and B antigen on RBC, His conclusion are
summarized as follows:
The antigen on the RBC determines the blood groups. For example, RBC with A
antigen on its surface makes the individual Type A
The corresponding antibody is never found in the individual’s serum, For example,
RBC with A antigen can not have anti-A in the serum
The opposite antibody is always present in the individuals’ serum. The antibodies of
the ABO system are usually IgM.
For example, an individual who is group A has the A antigen on his RBC; has no anti-A
in his serum, but always has anti-B in his serum
LANDSTEINER LAW
ABO Blood Groups

Blood Groups Antigens on RBC Naturally occurring Percentage in

surface antibody in serum American population
A A Anti-B 40%

B B Anti-A 10%

AB A and B None 5%

O None Anti-A 45%

Anti-B
ABO NOMENCLATURE
There are 3 systems of classification used in the ABO Blood Group System:

Landsteiner Jansky Moss

A I IV
B II II
AB II III
O IV II
GENETIC CONCEPTS
Bernstein first described the theory for the inheritance of the ABO blood groups in
1924.
Each parent contributes half of the genetic information to the child in hereditary units
called genes.
The genes are precisely organized like a string of beads on a strand of DNA known as
chromosome.
Each gene occupies a specific location on the chromosome called a locus/loci and at
each locus there may be several/different forms of genes, which are known as alleles.
The gene that codes for the A, B, or O blood type is located on the terminal portion of
the long arm of chromosome 9.
GENETIC CONCEPTS
The genotype refers to the notation of the actual genes inherited from the parents (½
from the mother and ½ from the father). The phenotype refers to the serologically
demonstrable antigens on the RBC membrane.
A genotype is homozygous if an offspring inherits identical alleles from both parents
(e.g. A from the mother and A from the father gives a homozygous genotype). A
genotype is heterozygous if the offspring inherits different alleles from the parents.
(e.g. A from the other and B from the father gives a heterozygous genotype).
An amorph (silent gene) is a gene that does not produce any detectable trait. The O
gene is an amorph since it does not produce detectable antigen on the RBC surface.
GENETIC CONCEPTS
A dominant gene is a gene that is always expressed in the offspring even though it is
only carried on one of the homologous chromosomes.
Example: A gene, B gene
A recessive gene is a gene that in the presence of its dominant gene does not express
itself. Expression occurs when it is inherited in the homozygous state.
Example: O gene

Chromosomes AA AO AB BB BO OO
Genotype AA AO AB BB BO OO
Phenotype A A AB B B O
INHERITANCE OF ABO BLOOD GROUPS
In 1930, Thomson suggested that 4 alleles are involved: A1, A2, B, and O. Each
offspring inherits two genes from the parents (one paternal and one maternal). Blood
groups are congenital
A method to determine the genotype of offspring is the box type diagram
Example 1: Father AA, Mother BB
A A
B AB AB
B AB AB

Genotypes: AB, AB, AB, AB

Phenotypes : 100% AB
INHERITANCE OF ABO BLOOD GROUPS
Example 2: Father BO, Mother AO
B O
A AB AO
O BO OO

Genotypes: AB= ¼ = 25%

BO= ¼ = 25%
AO= ¼ = 25%
OO= ¼ = 25%
Phenotypes : Type A = ¼ = 25%
Type B = ¼ = 25%
Type O = ¼ = 25%
Type AB = ¼ = 25%
FORMATION OF ABH ANTIGENS
The expression of the A and B genes depends on the action of H gene. Most people are
homozygous HH (others (Hh). The genotype hh is extremely rare and is referred to as
Bombay phenotype.
A precursor substance (mucopolysaccharide) is converted by the H gene to H
substance
The A or B gene convert part of the H substance to A or B antigen
Since O is amorphic, no conversion takes place, therefore, the H antigen is found in
highest concentration in Type O individuals
The order of greatest to least RBC reactivity with anti-H is O > A2 > B > A2B > A1 > A1B
FORMATION OF ABH ANTIGENS
ABH PRECURSORS
There are two potential precursor substances for ABH
antigens. Type I and Type II. Both are composed of 4
sugars: 2 molecules of D-galactose, one molecule of
glucose, and one molecule of N-acetylglucosamine. They
differ on the linkage of the terminal sugars.
Type I precursor has a terminal galactose (Gal) linked to
a subterminal N-aceytlglucosamine (Glc Nac) in a 1,3
linkage
Type II precursor has the same sugars combine in a 1,4
linkage
ABH antigens on RBC are derived from Type II chains
whereas the ABH antigens in plasma are made from
both Type I and Type II precursors
ABH ANTIGENS
Red cell ABH antigens consist of 80% glycoproteins and 20% glycolipids (derived from Type II
precursors)
The H antigen (the precursor of A and B antigen) is formed by the addition of fucose (Fuc) to the
terminal galactose (Gal) on either Type I or Type II precursors.
After fucose is attached to Type II precursors, the structure is called type II H antigen. Four types
of Type II H antigens are identified as:
H1 and H2 are simple straight chain glycolipids
H3 and H4 are branched chain glycolipids
A or B specificity is determined by the addition of monosaccharide to the terminal galactose of
the H substance
A antigen is formed by the addition of N-acetyl galactosamine to the terminal galactose of the
H substance
B antigen is formed by the addition of galactose to the terminal galactose of the H substance
CHEMICAL STRUCTURE OF THE ABH ANTIGENS
ENZYMES CODED BY THE A AND B GENES
The enzymes that add sugars to the terminal galactose of the H substance are coded
by the A and B genes

Gene Enzyme Sugar added

A 1,3 N acetyl galactosaminyl transferase N-acetyl galactosamine
B 1,3 galactosyl transferase D-galactose
H 1,2 fucosyl transferase Fucose
GENERAL CHARACTERISTICS OF ABO ANTIGENS
They can be demonstrated as early as second month of fetal life. Antigen A appears to
be weak at birth but at the age of one year, agglutinogens reach final strength
They persist throughout life unaltered, However, abnormal antigens may be found as
acquired characteristics in leukemia (weak A antigen) and cancer (abnormal secretion
of ABH substances)
They may be found in saliva, pancreatic secretions, gastric secretions of people who
are secretors
They may be found in bacteria and other species
GENERAL CHARACTERISTICS OF ABO ANTIBODIES
They are normally present at birth. If present at birth, they originated from the mother
through placental leakage during delivery
They developed 3-6 months after birth
They react better at room temperature (cold antibodies)
They occur in two forms:
Naturally occurring antibodies
Immune antibodies produced during incompatible transfusion or incompatible pregnancies
They are present in some animals and in plants as lectins. Lectins are plants or seed
extracts diluted to agglutinate specific human blood group antigens
They are present in low titer or even absent in cases of acquired and congenital
hypogammaglobulinemia and agammaglobulinemia
ABO ANTIBODIES
Anti-A, Anti-B
Produced by individuals who lack A and B antigens respectively
These antibodies are predominantly IgM
IgG anti-A and anti-B occur less frequently by Group O individuals
Anti-A,B
They are produced by the Group O phenotype. They are not simple mixture of anti-A
and anti-B but contain a third antibody that cross react with an antigen present on
both A and B RBC. This antigen is termed the compound AB or C antigen
ABO ANTIBODIES
Anti-A1
They are naturally occurring IgM antibodies produced by some A2 and A2B individuals
They usually have a low thermal range and for this reason are usually of no clinical
significance
Group O or Group A2 blood can be given to A2 individuals whose serum contains anti-A1 with a
thermal rage high enough to be clinically significant
Anti-H
They are naturally occurring auto antibody present in the serum of some A 1 and A,B
individuals
They have low thermal range and are seldom clinically significant
They occur as an IgG or IgM alloantibodies in the serum of Bombay individuals
The thermal range and ability to activate complement make anti-H clinically significant
Bombay individuals should be transfused only with blood from Bombay phenotype
individuals
ABO ANTIBODIES
Anti-A
Anti-B Anti-A1 Auto Anti-H Allo Anti-H
Anti-A,B
Clinically significant Yes sometimes No Yes
Low (common)
Thermal range Wide Low Wide
High (rare)
Transfusion No extra vascular Both extra and
Extra/intra vascular No
reaction Rare intravascular intravascular
Antibody class IgM, IgG IgM IgM IgM, IgG
HDN Yes No No Yes
H ANTIGEN AND ANTI-H
H antigen is the basic antigenic material of the ABO system. The quantitative level of the h
substance varies depending on the blood group
Group A and AB possess the smallest level of H substance on the cell membrane but as the
subgroups become weaker, more H material is found. This means that A 2B cells possess more
H substance than A1 cells
H antigen is detected by using anti-H antiserum derived from Ulex europeus
The reactions of A subgroups to anti-H is as follows:
Group Reactions to anti-H Group Reactions to anti-H
A1 Negative A4 (+++)
A2 (+) Ax (++++)
A3 (++) O (++++)
These reactions following the use of anti-H can be useful in the identification of the weaker
subgroups of A
THE BOMBAY PHENOTYPE
Individuals with the extremely rare Bombay phenotype (Oh) do not inherit the H gene (hh) and
are unable to produce H substance
Bombay individuals can inherit the A and B genes but because they lack the H substance, they
cannot make the A or B antigen
Unlike normal Group O RBC that has large amounts of H antigen, Bombay RBC lack H antigen
The children of Bombay individuals can express normal amounts of A or B antigen on their RBC
providing they inherit an H gene from one parent
Bombay individuals are compatible only with other Bombay individuals
Differences between True Type O bloods from Bombay blood
THE BOMBAY PHENOTYPE
Differences between True Type O bloods from Bombay blood
SECRETOR GENES (S )
The Se gene controls the presence of A, B and H antigens in saliva, sweat, tears,breat milk,
urine and semen
There are 3 genotypes: SeSe, Sese, sese. Se is dominant; therefore, SeSe and Sese are
secretors, i.e. these individuals secrete A, B, H antigens in secretions
In the cucasian population, 80% are secretors. The remaining 20% are non-secretors (sese)
An individual secretor status can be determined by testing for ABH substance in saliva
Other blood group substances are also secreted in plasma, and saliva (Chido, Lewis, Rodgers,
Sd9, I). Their presence in body fluids is not controlled by Se gene.
GROUP SPECIFIC SUBSTANCES FROM OTHER
SPECIES (WITEBSKY SUBSTANCES)
Blood group specific substances A can be extracted from hog stomach while blood group
specific substances B can be extracted from horse stomach
Witebsky play an important role in the production of commercial anti-sera. They can be used
to test the presence of immune isoagglutinins.
Natural isoagglutinins are neutralized by a small amount of group specific substances while
immune isoagglutinins remain active even if large amount of blood group specific substances
are added
ABO GROUPING
The ABO group of RBC is determined by two procedures:
Forward typing or direct typing or routine typing, cell typing
This involves determination of the unknown RBC agglutinogens by testing 2% RBC
saline suspension with known anti-sera (Anti-A and anti-B)
Backward typing, indirect typing, serum typing, back-up typing
This involves determination of the unknown antibodies in the serum using RBC
suspension of known types
Usually employed as a confirmatory test
ABO GROUPING
Cell Grouping (Forward typing, Routine typing)
Principle:
To perform cell grouping, the patient’s RBC are added to the following specific anti-sera:
Anti-A
Derived from Group B donors; reagent is colored blue because the serum is dyed with
trypan blue
Anti-B
Derived from Group A donors; reagent is colored yellow because the serum is dyed with
acriflavin
Anti-A,B
Derived from Group O donors
Used as a control for Anti-A and anti-B
Anti-A,B more effectively agglutinates
ABO GROUPING
Cell Grouping (Forward typing, Routine typing)
Pattern of Reactions: Recipients’ RBC plus
ABO Group Anti-A Anti-B Anti-A,B
Known A cells Known B cells O cells

A 4+ 0 4+

B 0 4+ 4+

AB 4+ 4+ 4+

O 0 0 O
ABO GROUPING
Serum Grouping
(Backward typing) Patient’s serum plus
Principle: patient serum ABO Group Anti-A Anti-B Anti-A,B
is added to known Group
A and Group B RBC. The Known A cells Known B cells O cells
presence of anti-A and/or
anti-B in the patient A 0 4+ O
serum will give a specific
pattern of agglutination B 4+ 0 O
Pattern of Reactions:
Agglutinates RBC with AB 0 0 O
weakened expression
(subgroups of A or B) O 4+ 4+ O
than the anti-A or anti-B
reagents
ABO SUBGROUPS – A PHENOTYPE
The A phenotype can be divided into subgroups:
A1 comprises approximately 80% of Group A individuals. Type A1 individuals make A
antigen from all type II H chains (H1, H2, H3, H4). Therefore, A1 individuals have more
A antigen per red cells than A2 individuals.
A2 comprises 20% of Group A individuals. They produce A antigen only from H1 and
H2 precursors. Approximately 3% of A2 individuals and 25% of A2B individuals
produce an antibody-designated anti-A1. This antibody reacts with A1 RBC but not
with A2 RBC.
A3 subgroup has an incidence of 1 in 1000 RBC. Some A3 cells are agglutinated
whereas others are not. This is termed mixed field reaction.
Rarer subgroups include Ax, Am, Aen, Ael and Afinn. It is important to identify blood
donors who have weak subgroups to avoid mislabeling their blood as Group O.
ABO SUBGROUPS – A PHENOTYPE
The major subgroups of A (A1 and A2) are differentiated by the use of:
Anti-A antiserum (obtained from serum of group B donors)
Absorbed anti-A1 antiserum (obtained serum of group B individuals that contain
anti-A and anti-A1. A2 cells are added to the serum of Group B individuals. Anti-A
reacts with A2 cells while anti-A1 do not react with A2 cells. After centrifugation, the
remaining filtrate contains anti-A1, which is now called absorbed anti-A1 typing sera
or anti-A1 typing sera)
Anti-A1 lectin(obtained from the extract of Dolichos biflorus seeds)
ABO SUBGROUPS – A PHENOTYPE
Patterns of reactions of A subgroups
A1 cells A2 cells
Anti-A + +
Absorbed anti-A1 + -
Anti-A1 lectin + -
(Dolichus biflorus)

The determination of subgroups of A is done to avoid misinterpretation as “O” instead

of A2 and B instead of A2B
ABO SUBGROUPS – B PHENOTYPE
Subgroups of B are quite rare and include B3, Bx, Bm, Bel.
To differentiate the subgroups of B1, anti-B lectin is employed which is an extract from
Banderaea simplicifolia
PROBLEMS IN BLOOD GROUPING
PROBLEMS ASSOCIATED WITH RBC SUSPENSION
Rouleaux Formation
It is the microscopic stacking of RBC in an overlapping position resembling like stack
of coins
It is due to increased serum proteins as in Waldenstrom’s macroglobulinemia or
multiple myeloma. Failure to wash cells leads to rouleaux formation.
The remedy involves repeating the test with saline washed RBC
To differentiate rouleaux formation and true agglutination, put a drop of saline to
the reaction mixture. If the clumping disperses then it is rouleaux formation
Mixture of cell types
It is due to transfusion of O blood to A and B individuals
To resolve the problem, check the patient’s history
PROBLEMS IN BLOOD GROUPING
PROBLEMS ASSOCIATED WITH RBC SUSPENSION
Chimerism
This is a phenomenon wherein two genetically different RBC populations are present in the
same person
Auto controls should be set up in these instances
Subgroups
A2 subgroups with or without anti-A1 poses common problem in blood grouping
Unusual phenotype such as Bombay blood. Bombay blood is tested with anti-H
Disease process
Leukemia and bacteremia (acquired B phenomenon) cause error in blood grouping
In bacterial contamination and septicemia, persons RBC may become agglutinable by all
sera except his own
PROBLEMS IN BLOOD GROUPING
PROBLEMS ASSOCIATED WITH SERUM
Room temperature reacting antibodies (or cold antibodies) such as antibodies in the following
blood group systems: H, I, M, N, P1, Lewis and anti-A1 in A2 or A2B individuals
Age
Infants under the age of 6 months do not usually possess the reciprocal antibody in their
plasma for the first few months of life (naturally occurring antibodies may not have
developed yet)
Infants may be typed by forward typing using infants RBC but not by backward or serum
typing
Elderly patients have decreased antibody production causing a problem in blood grouping
Compromised immune system or in hypogammaglobulinemia or in agammaglobulinemia
In these conditions, there is the reduction and/or absence of antibodies
ABO DISCREPANCIES
Technical errors
Inadequate identification of blood specimens, test tubes or slides
Cell suspension either too heavy or too light
Clerical error
A mix-up in samples
Missed observation of hemolysis
Failure to add reagents
Failure to follow manufacturer’s instructions
Uncalibrated centrifuge
Contaminated reagents
Warming during centrifugation
ABO DISCREPANCIES
ABO discrepancies are arbitrarily divided into four major categories:
Group I discrepancies are between forward and backward grouping because of weakly
reacting or missing antibodies
Newborns
Elderly patients
Patient with leukemia (CLL) demonstrating hypogammaglobulinemia
Patients with malignant lymphoma (may demonstrate hypogammaglobulinemia)
Patients using immunosuppressive drugs that yield hypogammaglobulinemia
Patient with congenital agammaglobulinemia and immunodeficiency states
Patients with bone marrow transplantation (patients develop hypogammaglobulinemia
from therapy and start producing a different red cell population from the transplanted
bone marrow)
ABO DISCREPANCIES
ABO discrepancies are arbitrarily divided into four major categories:
Group II discrepancies are between forward and reverse groupings because of weakly
reacting or missing antigens
Subgroups of A and B antigen
Leukemia (may yield weakened A and B antigens)
Hodgkin’s disease (may mimic the depression if antigen in leukemia)
Excess amounts of blood group specific soluble substances (BGSS) present in plasma due
to carcinoma of the stomach and pancreas
Acquired B phenotype often associated with intestinal obstruction or malignancy of the
stomach and intestines
ABO DISCREPANCIES
ABO discrepancies are arbitrarily divided into four major categories:
Group III discrepancies are between forward and reverse groupings caused by proteins or
plasma abnormalities and result in rouleaux formation or pseudoagglutination
Elevated levels of globulin as in multiple myeloma, Waldenstrom’s macroglobulinemia and
advanced cases of Hodgkin’s lymphomas
Elevated levels of fibrinogen
Plasma expanders such as dextram and polyvinylpyrrolidone (PVP)
Wharton’s jelly
ABO DISCREPANCIES
ABO discrepancies are arbitrarily divided into four major categories:
Group IV discrepancies are between forward and backward grouping
Polyagglutination
Cold reactive antibodies (allo and auto)
Warm autoantibodies
Unexpected ABO isoagglutinins