Fundamentals of
Immunohematology
Lecture 1
Historical Perspectives:
Pope Innocent VII 1492
Vein to vein transfusions
Karl Landsteiner discovered ABO blood groups 1900
1915 anticoagulants-citrate-phosphate-dextrose
WWII & American Red Cross
Frequent transfusions /circulatory overload- need
for components instead of whole blood
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BY-SA.
The Donation Process
1. Educational materials
2. Donor health history questionnaire
3. Abbreviated physical examination
RBC Biology and Preservation
RBC shape is a biconcave disc
RBC composition- 52% protein, 40% lipid, and
8% carbohydrate
Three areas of RBC biology are necessary for normal
erythrocyte survival and function:
1. normal chemical composition and membrane
structure.
2. Hemoglobin structure and function
3. RBC metabolism
Defects in any of these will shorten
the RBC survival time.
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BY-SA.
Normal length of survival of RBCs is 120 days in circulation.
RBC metabolism – anaerobic glycolytic pathway, pentose phosphate
pathway, methemoglobin reductase pathway, and the LueberingRapoport pathway.
Hemoglobin's function is to transport O2 to the tissues and CO2
excretion.
The amount of 2,3, DPG within RBCs has a significant effect on the
affinity of hemoglobin for oxygen and therefore affects how well RBCs
function post-transfusion.
Oxygen dissociation curve- shift to the left,
shift to the right​
Hemoglobin's function-transport O2 to
tissues and CO2 excretion.
Shift to the right-decrease in hemoglobinoxygen affinity. Increased levels of 2,3,DPG.
Shift to the left- increases hemoglobinoxygen affinity. Decreased levels of
2,3,DPG.
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Anticoagulant solution CPDA-1 extends shelf
life to 35 days at 1.0-6.0 C
Additives can extend to 42 days from
collection.
RBC freezing for rare blood types and
autologous units.
Two concentrations acceptable: highconcentration glycerol 40% weight by volume,
low-concentration glycerol 20% weight by
volume. Frozen RBCs must be thawed and
washed prior to transfusion.
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Platelet Preservation:
Platelets help to form the hemostatic plug
Platelet apheresis minimum of 3.0 x 10 11th in
platelets
Gently agitated at 20-24C (room temperature)
Spontaneous hemorrhage may occur when platelet
count falls below 10,000.
Stored at room temperature and have a 5 day shelf
life. Two main reasons only 5 days- bacterial
contamination at incubation of 22C more likely and
loss of platelet quality during storage.
Absence of platelet swirling is associated with loss of
membrane integrity
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Storage Lesion
RBCs-loss of ATP which causes
loss of membrane
deformability and more fragile
with age. Leads to release of free
hemoglobin and K.
Platelets- loss of platelet quality
and viability due to loss of
membrane integrity. A pH of <6.2
is associated with loss of platelet
viability.
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licensed under CC BY.
• Basic Genetics
• Gene: a segment of deoxyribonucleic acid that encodes for a
particular protein
• Trait: A genetically determined characteristic or condition
• Gene locus: The specific location on chromosomes at which genes
are arranged
• Allele: Alternative forms of a gene
• Chromosome: Gene-carrying structures that are visible
during nuclear division in the nucleus of the cell
• Each living cell has a characteristic set of chromosomes in the
nucleus. These chromosomes are made up of nucleic acids and associated
proteins that carry genes.
• Each chromosome has 2 arms that are joined at a central
constriction called the centromere.
• The chromosomal arms are different lengths; the short arm (p) and
the long arm (q).
• Carrier: an individual who carries one gene for a recessive trait and one
normal gene
• Homologous chromosome: a pair of chromosomes in which males and
females carry equivalent genes
• Autosome: Any chromosome that is not a sex chromosome
• Sex chromosome: Chromosome that determines gender
Mendel’s laws of inheritance –law of
independent segregation. Each gene is
passed on to the next generation on its
own.
Mendel’s second law- law
of independant assortment. Genes for
different traits are inherited separately from
each other.
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licensed under CC BY-SA.
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Homozygous -identical alleles for a given locus or present on both
chromosomes. (double dose)
Heterozygous -different alleles are present at a given locus.
Hemizygous -only a single copy of an allele is present instead of the
customary two copies.
Antigens that are encoded by alleles at the same locus are said to be
antithetical.
Inheritance of Genetic Traits
A genetic trait is the observed expression of one or more genes.
Autosomal Dominant Inheritance- trait is always expressed it appears
in every generation and occurs in equal frequency in males and
females.
Autosomal Codominant Inheritance- when two different alleles are
present (heterozygous) the products of both alleles are expressed.
Autosomal recessive inheritance expressed only in a person who is
homozygous for the allele Ann has inherited the recessive allele from
both parents.
Sex linked inheritance a trait that is sex linked is one that is
encoded by a gene located on the X or Y chromosome.
In females the inheritance of X linked genes can be dominant or
recessive.
Males are hemizygous for genes on the X or Y chromosome because
there is only one chromosome present.
Thus, an X linked trait that is recessive in females is expressed by all
males who carry the gene for the trait. With X linked
inheritance that trade is never transmitted from father to son.
Sex linked dominant inheritance- males pass to all daughters, who
then all express the trait. Heterozygous females pass the trait to 50% of
their children. Homozygous females will pass the trait to all
of their children. Ex:Xga
Sex linked recessive inheritance is carried but not expressed by a heterozygous
female. A male inherits the trait from his mother who is usually a carrier. An
unaffected male transmits the trait to all of his daughters, who in turn transmit
the trait to half of their sons. The prevalence of the expression of an X
linked recessive trait is much higher in males than in females. ex: XK in
McLeod phenotype.
Click to add text
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Fundamentals of Immunology
The majority of blood bank testing is focused on the prevention, detection,
and identification of blood group antibodies and on the typing of RBC
antigens. Antigen characteristics and host factors have an impact on the
immune response. The detection of alloantibodies or
autoantibodies in routine testing procedures is one of the utmost
importance in providing compatible blood to patients. This detection is
dependent upon several factors, including binding forces between antigens
and antibodies, properties of the antibody itself, and individual host
characteristics. Antigen-antibody reactions are influenced by a number
of factors, including distance, antigen-antibody
ratio, pH, temperature, and immunoglobulin type.
Innate immune response consist of physical barriers, biochemical
effectors and, an immune cells. The first step of the innate defenses
external including skin and enzymes presence on the skin's surface. The
second line of innate defenses internal an can recognize common
invaders with a nonspecific response.
Cellular immunity is mediated by various IS cells such as macrophages, T
cells, and dendritic cells.
Humoral immunity consists of fluid parts of the IS, such as antibodies and
complement components found in plasma, saliva, and other secretions.
Antibodies are also called
immunoglobulins, immune
because of their function and
globulin because they are a type of
globular soluble protein.
They are found in the gamma
globulin portion of the plasma or
serum. The function of the
antibody is to bond afford
molecules called antigens. Only
one antibody reacts with one
antigen, or one part of a complex
antigen.
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licensed under CC BY-SA.
Innate or natural immunity is the immediate line of immune defense
and is present at birth.
1st line (innate)- skin, mucous membranes, cilia, cough reflex,
secretions, low ph of vag and stomach.
2nd line (innate)-phagocytic cells (macrophages, monocytes, PMNsgranulocytes, NK cells, complement- alternate pathway, cytokinesinterferons and interleukins, acute inflammatory reaction.
Acquired or Adaptive immunity- specific, evolved immune system
The acquired IS uses antibodies as specific immune effectors and has
memory.
There are two major arms of the acquired system:
Humoral- mediated by B cells and antibody production
Cellular – mediated by T cells and lymphokines
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•T lymphocytes matures in the thymus gland and is
responsible for making cytokines and destroying
virally infected host cells​
•T cell mediated immunity is involved in response
against fungal and viral infections, intracellular
parasites, tissue grafts, and tumors​
•B lymphocytes matures in the bone marrowand
when stimulated by an antigen, evolve into plasma
cells that secrete antibody​
•Memory B cells have antibody on their surface
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T and B cells communicate with each other and are both
necessary for antibody production. B cells undergo gene
rearrangement in order to have the correct antibody made so
that they can react with the correct antigen. T cells also have
receptors that undergo gene rearrangement. The receptors of
the cell membranes of T&B lymphocytes allow them to
recognize foreign substances. Self vs non-self
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Mature B cells are called plasma cells and they bind to antigens in a specific
manner. The images are usually insoluble form in plasma. Memory B cells have
antibody on their surfaces. Plasma cells are antibody factories that make large
amounts of one specific type of antibody in a soluble form that remains in the
circulation in the plasma, body secretions and the lymphatics. Binding of energy
and by antibody brings about opsonization which aids in the direct killing of the
pathogens by cell lysis.
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T cell-mediated immunity is involved in the response
against fungal and viral infections, intracellular
parasites, tissue grafts, and tumors. T cell receptors
do not recognize foreign antigen on their own they
require help in the form of cell membrane proteins
known as the major histocompatibility complex
molecules (MHC).
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Two major classes of MHC genes and antigens:
MHC class I – on nucleated cells, code for HLA-A, HLA-B, HLA-C
antigens
MHC class II- on most antigen presenting cells, code for HLA-DR,
HLA-DQ, HLA-DC
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It usually takes days to months from exposure to form an initial antibody.
This is called the lag phase or preseroconversion window. It is during this
time the antibody cannot be detected. The primary antibodies are IgM
antibodies.
After the antigen is cleared memory cells are stored in the immune organs
of the host when the same antigen is encountered again the memory cells
are activated and produce a stronger and more rapid response. IgG
antibodies are formed during the secondary response intermating great
quantities. Secondary antibodies can usually be measured within one to
two days.
Immune Maturation
• Takes days to months for immune response to make initial antibody
• The lag phase-latency, preseroconversion or window period
• After antigen is cleared, memory cells are stored
• When the same antigen is encountered again the immune response
is quicker and stronger. This is called the secondary
(memory) response.
Immunoglobulin Structure
• An antibody is a tetramer of two identical heavy chains and two
identical light chains
• Each heavy and light chain contain a variable region- which varies
from antibody to antibody and binds antigen- and a constant region
• Human antibodies consist of two light chain families and any given
antibody has either two identical kappa or two identical lambda light
chains
The Fab fragment consists of the heavy-and
the light- chain variable regions, the lightchain constant region, and one heavy-chain
constant region domain. The Fab fragment
binds antigen but does not activate effector
mechanisms.
The Fc fragment, which consists only of
heavy-chain constant regions, activates
effector mechanisms, which allows for the
destruction of the antibody target. The Fc
constant regions differ based on antibody
isotype and subclass
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There are five different antibody
isotypes which are determined by the
constant region of the heavy chain.
These include IgM, IgG, IgE, IgA,
and IgD.
Antibody isotypes can differ in the
number of antigen-binding sites per
molecule
The number of antigen-binding sites
per antibody affects the binding avidity
for the antigen.
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under CC BY-SA.
IgM antibodies are capable of
activating complement by
changing their structure after
antigen binding; they are
known to
cause hemolysis during
transfusion reactions​
IgM antibodies are capable of
activating complement by
changing their structure after
antigen binding; they are
known to cause hemolysis
during transfusion reactions.
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BY-SA-NC.
IgG antibodies are divided into four subclasses: IgG1, IgG2, IgG3 and IgG4.
Each subclass has a different constant region and interacts differently with Fc
receptors on phagocytes. Some are capable of complement activation.
IgG1 and IgG3 are the most effective in activating complement.
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Complement system- has 3 major roles:
final lysis of abnormal and pathogenic cells
via the binding of antibody,
opsonization and phagocytosis, and
mediation of inflammation.
Classical pathway and Alternate pathway
of Complement system
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1. Classical Pathway is
initiated when antibody
binds to antigen.
C1 binds to Fc fragment
of IgM, IgG1 or IgG3
subclass antibody.
Complement
2. Alternative pathway is
activated by surface
contact with complex
molecules and artificial
surfaces.
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3. Lectin pathway is activated by attachment to MBL to microbes.
All three methods of activation lead to a final common pathway for
complement activation and membrane attack complex (MAC)
formation.
•Lymphokines are powerful molecules that include
cytokines and chemokines
•Antibodies are also called immunoglobulins and are found
in the gamma globulin portion of the plasma or serum​
•The function of the antibody is to bind to foreign
molecules called antigens. Only one antibody reacts with
one antigen or one part of a complex antigen. Lock and key
mechanism.
• Alloantibody- antibody produced in an individual against red blood cell
antigens of another individual
• Autoantibody- antibody produced against the individuals own
red blood cell antigens
• Antigen and antibody reactions are influenced by a number
of factors, including distance, antigen-antibody ratio, pH, temperature,
and immunoglobulin type
The immune response is initiated by the presentation of an antigen
(initiates formation of and reacts with an antibody) or immunogen
(initiates an immune response).
Properties such as size, complexity, conformation, charge, accessibility
solubility, digestibility, and biochemical composition influence the amount
and type of immune response.
RBC antigens are diverse in structure and composition and may be
proteins (such as Rh, M, and N blood group substances) or glycolipids
(such as ABH, Lewis, Ii, and P blood group substances). Human leukocyte
antigens (HLAs) are glycoproteins.
Most naturally occurring antibodies are IgM cold agglutinins. These
react best at room temperature or lower, activate complement, and
may be hemolytic when active at 37C. Ex: ABO, Hh, Ii, Lewis, MN,
and P blood group systems
Most RBC antibodies are IgG that react best at 37C and require the
use of antihuman globulin sera for detection. The most common and
clinically significant of these are Rh, Kell, Duffy, Kidd, and Ss blood
group systems.
Naturally occurring
anti-A and anti-B
antibodies are
routinely detected
in human serum and
depend on the
blood type of the
individual.
These naturally
occurring antibodies
or isoagglutinins are
significant and are
useful in blood
typing.
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BY-SA.
All other antibodies directed against RBC antigens are considered
unexpected and must be detected and identified before blood can be
safely transfused, no matter their reaction strength or profile.
Antibodies can be either alloreactive or autoreactive. Alloantibodies are
produced after exposure to genetically different (non-self) antigens, such
as RBC antigens from transfusion.
Titers of alloantibodies may fall and be undetectable. If these indivduals
are transfused with the immunizing antigen again they will make a
stronger immune response which can cause a severe or possibly fatal
transfusion reaction. This is why the previous history of any patient is a
critical part of the testing.
Intermolecular binding forces- hydrogen bonding,
electrostatic forces, van der Waals forces, and
hydrophobic bonds are involved in antigen-antibody
binding reactions.
Affinity-strength of a single antigen-antibody bond
produced by attractive and repulsive forces.
Avidity- binding strength of a multivalent antigen
with antisera produced from an immunized
individual.
High-titier, low-avidity antibodes exhibit low antigen
binding capacity but still show reactivity at high
serum dilutions.
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Blood samples 4 testing
The anticoagulant EDTA (ethylenediaminetetraacetic acid) [ethylenediamine-tetra-acetic-acid] at a ratio of 2mg to 1ml, is most commonly
used. EDTA inhibits complement activation by binding calcium and to
a lesser extent magnesium.
Another anticoagulant that is sometimes uses sodium heparin which
inhibits the cleavage of C4. Plasma samples are often preferred
over serum for testing because they lack fibrin strands which can cause
false positives.
Serum can be used for testing.
Centrifugation- enhance agglutination reactions because it decreases
reaction time by increasing gravitational forces on the reactants and
bringing reactants closer together.
High speed centrifugation is the most efficient method used in blood
banking.
Antigen-antibody ratio:
Prozone-excess unbound immunoglobulin
Postzone- surplus antigen
Both prozone and postzone can yield a false negative result.
PH- between 6.5 and 7.5
Temperature-IgM antibodies usually react optimally
at ambient temperature or below 22C at the immediate spin (IS) phase,
IgG usually require 37C incubation and react optimally at the antihuman
globulin (AHG) phase.
Enhancement media reduces the zeta potential of RBC membranes.
The net negative charge surrounding RBCs is part of the force that repels
RBCs from each other and is due to sialic acid molecules on the surface
of the RBCs.
Albumin 22% and PEG are colloids that increase the dielectric constant
which then reduces the zeta potential of the RBC.
Low ionic strength solutions (LISS) decreases the ionic strength of a
reaction medium which reduces the zeta potential.
Enzymes are protein molecules that function by altering reaction conditions
and bringing about changes in other molecules without being changed
themselves.
Ex: ficin (from fig plants), papain (from papaya), trypsin (from pig stomach)
and bromelin (from pineapple).
Enzymes enhance Rh, Kidd, P1, Lewis, and I antigens.
Enzymes destroy Fya, Fyb, M, N, and S antigens.
Dithiothreitol (DTT) and beta-2-mercaptoethanol (2-ME) are thiolreducing agents that break the disulfide bonds of the J chain of the IgM
molecule but leave the IgG molecule intact.​
ZZAP is a thiol reagent plus a proteolytic enzyme. It causes dissociation
of IgG molecules from the surface of sensitized RBCs and alters the
surface antigens of RBCs.
Flow cytometry makes use of antibodies that are tagged with a
fluorescent dye. Fluorescence occurs when the compound absorbs light
energy of one wavelength and emits light of a different wavelength.
End of Lecture 1
Read Chapters 1-4 and complete quiz.