REVIEW OF GENETICS

GENETICS

• It is the study of inheritance – the transmission of

characteristics from parents to offspring

• Is the scientific study of heredity

• Study how traits and disease are passed from generation

to generation
DEFINITION OF TERMS

• Gene – the fundamental units of heredity and the basic

structural and functional units of genetics

• Mendel call genes as “factors”

• Determine the traits that can be present but not expressed

DEFINITION OF TERMS

• Dominant traits – the traits expressed in the F1(heterozygous)

condition

• Recessive traits – The trait unexpressed in the F1 but re-

expressed in some members of the F2 generation
DEFINITION OF TERMS

• Phenotype – the observable properties of an organisms

• Genotype – the specific genetic constitution of an organism

• Segregation – the separation of members of a gene pair from

each other during gamete formation
 DEFINITION OF TERMS

• Allele – one of the possible alternative forms of a gene, usually

distinguished from other alleles by its phenotypic effects

• Homozygous – having identical alleles for one or more genes

• Heterozygous – carrying two different alleles for one or more

genes
AUTOSOMAL RECESSIVE
GREGOR MENDEL
• An Austrian monk, experimented with pea plants to study physical
traits and how they are inherited.

Mendelian Laws:
•Law of Independent Segregation

•Law of Independent Assortment

 GREGOR MENDEL

• Studied the inheritance of shape

• Experimental Specimen: Peas
• Smooth shaped peas crossed with wrinkled
shaped peas
LAW OF INDEPENDENT SEGREGATION

• Gene exist in pairs and there must be only one gene to exist, to prevent gene
number in doubling in each succeeding generation

• Individual traits are inherited separately from one another

• Alleles of genes have no permanent effect on each other when present in the
same organism, but segregate unchanged by passing into different gametes
LAW OF INDEPENDENT ASSORTMENT

• Genes for different traits are inherited separately from each other; allows possible
combinations of genes to the offspring

• Members of one gene pair separate from one another independently of the member of
other gene pairs

• The random distribution of alleles into gametes during meiosis

• Smooth – dominant; Wrinkled – recessive

• Yellow – dominant; green - recessive
VARIATIONS ON A THEME BY MENDEL

1. Incomplete dominance
 Expression of a phenotype that is intermediate to those of the parents
2. Codominant alleles
 Full phenotypic expression of both members of a gene pair in the
heterozygous condition
3. Genes have more than two alleles
INCOMPLETE DOMINANCE

R2R2
R1R1 (WHITE)
(RED)

R1R2
(PINK)
CODOMINANT ALLELES
GENES HAVE MORE THAN TWO ALLELES
CELL DIVISION

• Dividing cells are needed in order to study chromosomes

• Cytogenetic abnormalities result from errors in cell division

2 types:
• MITOSIS – Division of somatic cells
• MEIOSIS - division that occurs only on gametes (sex cells)
IMMUNOLOGY IN BLOOD BANKING
 IMMUNE SYSTEM:

• Immunology is both the study of the human immune system and

the field of medicine that treats diseases of the immune system

• Immunity is a process by which a host organism protects itself

from attacks by external and internal agents
 MAJOR MECHANISM OF IMMUNE SYSTEM

Innate or Natural immunity

Primary lines of defense
Non-specific
• Natural – present at birth
• Immediately available
• Maybe physical, biochemical, mechanical or a combination of
defense mechanism
Does not alter on repeated exposure to any specific Ag
 MAJOR MECHANISM OF IMMUNE SYSTEM
Acquired or Adaptive Immunity
Supplements protection provided by innate immunity
Later evolutionary development – seen only in vertebrates
Specific
• Specialized
• Acquired by contact with a specific foreign substance
• Initial contact with foreign substance triggers synthesis of
specialized Ab proteins resulting in reactivity to that particular
foreign substance
IMMUNOGLOBULINS:
• Also called Antibody
• Complex protein produced by plasma Ig are classified according to the
cells with specificity to Ag’s that molecular structure of their heavy
stimulate their production chains:
• A specific self protein produced by IgA
the host in response to specific, IgD
foreign, non self protein or other
IgE
complex molecule not tolerated by
the host IgG
IgM
IMMUNOGLOBULINS:
Immuno- function

Globulin- type of globular soluble protein found in the gamma globulin portion of serum
or plasma

Most important factor in Humoral immunity

Classified according to the molecular structure of their heavy chains

IgG is the most concentrated in serum – 80% of the total serum
IgA – 13% it is the major Ig found in body secretions
IgM – 6%
IgD – 1%
IgE – less than 1%
CHARACTERISTIC OF ANTIGEN:
• Can initiate formation of and react with an antibody
• Usually detected in blood bank
• Immunogen – can initiate an immune response to the immune system
• Hapten
 an immunogen that has a MW of < 10,000 Daltons
do not elicit an immune response; molecules that are too small cannot stimulate
antibody production
when coupled with carrier protein having a MW > 10,000 daltons can produce a
reaction
SIGNIFICANT ANTIBODIES IN BLOOD BANK

• IgG, IgM and IgA are the most significant Ab encountered in BB

• Clinically significant Abs reacts at 37 degree Celsius – IgG isotype
capable of destroying transfused Ag-positive RBC
• Naturally-occurring Abs reacts at 22 degree Celsius – IgM isotype
produced in response to naturally occurring antigens (intestinal
flora and pollen grains)
SIGNIFICANT ANTIBODIES IN BLOOD BANK
 IgG
 causes anemia and transfusion reactions, most IgG are clinically significant and are immune antibodies
 IgM
 Problem with IgM Ab is that they can interfere with the detection of clinically significant IgG Ab by
masking their reactivity
 Unlike IgG, IgM exists in both monomeric and polymeric forms
 Most IgM are naturally occurring
 IgA
 30% of Anti-A and Anti-B is of the IgA class (the remaining percentage are for IgG and IgM)
 Anti-IgA can cause severe problems if transfused in plasma products to patients who are deficient in IgA
result to fatal anaphylaxis
 IgA- can also increase the effect of IgG induced RBC hemolysis
 IgE
 found in monomeric form in trace concentration in serum about 0.004% of total Ig and is important in
allergic reactions, causes transfusion reaction by release of histamines (Ex. Urticaria)
 IgD
 least significant for blood banking; present in <1%- appears to have functions that deal primarily with
maturation of B cells into plasma cells, regulatory role during B cell differentiation and antibody
production
 CHARACTERISTIC OF BLOOD GROUP ANTIBODIES:

POLYCLONAL AB MONOCLONAL AB
Antibodies produced in response to a Isolated individual B cells from a
single antigen with more than one polyclonal population and propagating
epitope them in cell culture with hybridoma
(single epitope)
Vary in antibody concentration from Contains Ab from a single type of B cell
person to person and animal to animal

Not specific Highly specific, well characterized and

uniformly reactive
 CHARACTERISTIC OF BLOOD GROUP ANTIBODIES:
NATURALLY OCCURING IMMUNE ANTIBODIES

Ab’s found in the serum of individuals who who have never Found in the serum of an individual who has been exposed
been previously exposed to RBC antigens to certain antigens (blood transfusion, pregnancy)

Probably produced in response to substances in the IgG antibodies - reacts best at 37degree Celsius and require
environment that resembles RBC antigens the use of AHG (Coombs sera) for detection

• IgM cold agglutinins • Rh, Kell, Duffy, Kidd and Ss blood group system
 react best at RT or lower, activates complement,
may be hemolytic when active at 37 degree C
 All individuals except newborn babies have
naturally acquired T cell independent, IgM abs in
their plasma that is directed against any ABO and H
antigens that is lacking to them

• ABH, Hh, Ii, Lewis, MN and P blood group system

 CHARACTERISTIC OF BLOOD GROUP ANTIBODIES:
ALLOANTIBODIES AUTOANTIBODIES

Produced after exposure to genetically different or non-self Produced in response to self antigens
RBC Ag’s after transfusion (foreign antigens)

 Transfused components can elicit an immune response  Patients with autoimmune disease.
 Comes from alloantigens (present only in some  Can cause reaction to the recipient if they have a
individuals) specificity that is common to the transfused blood. Some
 One major problem in BB is when alloantibodies are no do not have detectable specificity are referred to as pan-
longer detectable in the patients serum or plasma, when polyagglutinins
transfused with immunizing Ag again; will make much
stronger immune response
React at different temperatures: cold or warm React at different temperatures: cold or warm

Not present in the recipient Can be removed by special adsorption and elution technique

Detected through antibody screening and identification Detected through antibody screening and identification
Positive in DAT and autocontrol
ZETA POTENTIAL
 The net negative charge surrounding RBCs (and most other human cells) in a cationic media is
part of the force that repels RBCs from each other and is due to sialic acid molecules on the
surface of RBCs
 Most acids have a negative charge, and the large concentration of these molecules on RBCs
creates a “zone” of negative charge around the RBC
 This zone is protective and keeps RBCs from adhering to each other in the peripheral blood. A
potential is created because of the ionic cloud of cations (positively charged ions) that are
attracted to the zone of negative charges on the RBC membrane
 Reducing the zeta potential allows the more positively charged antibodies to get closer to the
negatively charged RBCs and therefore increases RBC agglutination by IgG molecules
 POTENTIATORS/ENHCANCEMENT MEDIA
Reagent Action Procedure Type of Ab ID
AHG Crosslinks sensitized 1. DAT 1.Polyspecific: anti-
cells, resulting in visible 2. IAT IgG+ anti-complement
(antihuman- agglutination (C3d)
globulin) 2. IgG monospecific:
anti-IgG only

22% Albumin Causes Agglutination by Incubation @ 37C IgG

adjusting zeta potential for 15-60 min: cells
between RBCs washing prior to IAT

LISS (Low ionic Causes RBCs to take up Incubation@ 37dC IgG

Ab more rapidly for 5-15min; cell
strength saline) washing prior IAT
 POTENTIATORS/ENHANCEMENT MEDIA
Reagent Action Procedure Type of Ab ID
PEG (Polyethylene Increases test Incubation @ 37C IgG
glycol) sensitivity for 10-30min; cell
washing before IAT
Enzymes Reduces RBC 1. One step: Destroys Fya, Fyb,
Ficin (isolated from surface charge; enzymes added MNS; enhances
fig plants)
Papain (from destroys or directly to reactivity to Rh,
papaya) depresses some serum/RBC Kidd, P1, Lewis and
Trypsin (from pig
stomach)
RBC antigens; mixture I antibodies
Bromelin (from enhances other 2. Two step: RBC
pineapple) RBC Ag pretreated with
enzymes before
addition of
serum
POTENTIATORS/ENHANCEMENT MEDIA
Albumin and PEG are under colloids or proteins-its function is to enhance/increase the
dielectric constant (measure of electrical conductivity) and reduce zeta potential. PEG is
more effective in detection of weak antibodies and reduction in false positive or
nonspecific reactions
Enzymes are very effective in cases in which there are multiple antibodies present in a
sample. Also enzymes removes hydrophilic glycoproteins from the RBC membrane
causing the membrane to become more hydrophobic which would allow RBC to come
closer together and antibodies may no longer be subject to physical obstruction and
steric hindrance from reacting with RBC

Direct antiglobulin test (DAT): AHG added to washed RBC’s

Indirect antiglobulin test (IAT): serum+screen cells; Incubation @ 37 degree Celsius time
depends upon the additive used; cell washing; before addition of AHG
 MONOCLONAL VS. POLYCLONAL REAGENTS
Polyclonal Monoclonal
Produced by immunizing donors with Made by hybridoma technology with
small amount of RBCs for an antigen that spleen lymphocyte from immunized mice
they lack and then collecting the serum that are fused with rapidly proliferating
and isolating the antibodies against that myeloma cells
antigen
Polyclonal reagents are directed against Monoclonal reagents are directed against
multiple epitopes specific epitopes

Uses Human for reagent preparation (AHG) Do not use a human source
POLYCLONAL ANTIBODY PRODUCTION

• Involves hyperimmunization of rabbit by injecting purified

serum human IgG and C3d (antigens)
• The reagent should consist of a pool of rabbit anti-human IgG and
mouse monoclonal anti C3b and anti C3d
• Also refered to as Broad spectrum coombs reagent
 MONOCLONAL ANTIBODY PRODUCTION

 Georges Kohler and Cesar Milstein were awarded the Nobel Prize
in 1984 for their work in monoclonal antibody production by
Hybridoma Technology
 Monoclonal Antibody – Very specific derived from a single
antibody-producing cell that has been cloned or duplicated
 Hybridoma – A cell line resulting from the fusion of myeloma cell
and a plasma cell. These can be maintained in tissue culture
indefinitely and can produce a very specific type of antibody known
as monoclonal antibody
•
• Hybridoma is produced as follows:
• 1. Mouse is immunized with a certain antigen and after a time, spleen cells are harvested.
• 2. Spleen cells are combined with myeloma cells in the presence of polyethylene glycol
(PEG), a surfactant. The PEG brings about fusion of plasma cells with myeloma cells, producing
a hybridoma
• 3. After fusion, cells are placed in culture using HAT Medium (medium containing
hypoxanthine, aminopterin, thymidine). The medium ALLOWS Hybridoma cells to grow
selectively in tissue culture. MYELOMA CELLS (cancerous plasma cells) CANNOT grow in this
medium because of the myeloma cell line is deficient in the required enzymes hypoxanthine
guanine phosphoribosyl transferase (HGPRT) and thymidine kinase. Also, the growth of
myeloma cells is blocked by the presence of aminopterin
• 4. Myeloma cells and even normal B cells die out, leaving behind the fused hybridoma cells
• 5. Remaining hybridoma cells are diluted out and placed in microtiter wells where they are
allowed to grow. Each well containing one clone is then screened for the presence of
desired antibody by removing the supernatant
• 6. Once identified, a hybridoma is capable of being maintained in cell culture indefinitely
and it produces a ready supply of monoclonal antibody that reacts with a single epitope
Mouse plasma cell + myeloma cell = hybridoma cell
FACTORS ANTIGEN-ANTIBODY REACTIONS:
Intermolecular binding forces
 Ionic bonds, Hydrogen bonds, Hydrophobic bonds, Van der Waals forces
Antibody properties
 AFFINITY: initial force of interaction that exists between a single fab site on an
antibody molecule and single epitope or determinant site on the corresponding
antigen
 AVIDITY: sum/strength of all attractive forces between an antigen and an antibody
Host factors
 nutritional status, hormones, genetic inheritance, age, race, sex, physical activity,
and environmental exposure, occurrence of disease or injury
Tolerance
DETECTION OF RBC-ANTIGEN ANTIBODY REACTIONS
AGGLUTINATION
 process by which particulate antigens such as cells aggregate to form large complexes
when a specific antibody is present
 HEMAGGLUTINATION  Antigen is found on red cells
 HAS TWO PHASES:
 Sensitization  antigen binding to the antibody (epitopes on the RBC membrane
binds with the Fab region of the antibodies), no visible agglutination
 Lattice-formation  multiple antigen-antibody bridges RBC antigens and
antibodies is formed; with visible agglutination
 HEMOLYSIS
 Disruption of the red blood cell membrane and the subsequent release of hemoglobin into the suspending
medium or plasma
PHASES OF AGGLUTINATION
AGGLUTINATION GRADING
NEGATIVE AGGLUTINATION VS HEMOLYSIS
END