Blood Banking-1-1

LECTURE NOTES BY: JOASH-EVANGEL Z.
CAMPOS, RMT, MLS (ASCPi)
HISTORY OF TRANSFUSION MEDICINE ▪ “And he said, What hast thou

done? The voice of thy
Blood Facts: brother’s blood crieth unto
- Source of life (transports oxygen, proteins me from the ground. And
and other essential nutrients) now art thou cursed from the
- An average person of 73kg = 5L of blood earth, which hath opened
(needs 1,200,000 mosquitoes to drain all her mouth to receive thy
blood in the body) brother’s blood from thy
- 8% of body weight is blood hand.”
- Coconut water can be a substitute of blood o Leviticus 7:11
plasma ▪ “For the life of all flesh is the
o Low Sodium, High Potassium (same blood thereof…”
as fluid found in RBC)
o Loaded with Calcium and
Magnesium
o More Acidic 1ST TRANSFUSION MYTH (1ST Account of B.T.)
- Mosquitoes prefer Blood Type O 1492: Pope Innocent VII
- One pint of blood can save up to 3 lives - Drinkard (1870):
- 3 components: o “…a Jewish daring innovator, whose
(1) Plasma name has not come down to us in
(2) RBC memory of his deed, proposed to find
(3) Platelets the pontiff a fountain of jouvenance
in the blood of three youths who died
Blood in History: as martyrs to their own devotion and
- Ancient Civilization believes in the potential the practitioners zeal.”
of Blood - Said to have received, at the behest of a
o China (1000 BC): Jewish physician, a transfusion of the blood of
▪ Soul is contained in the blood three 10 y.o. boys, each of whom was paid a
o Egypt: ducat and all of whom died.
▪ Swimming or bathing in a o Probably the blood was drawn but was
pool of blood for their health intended to be taken orally. Indeed,
o Rome: Pliny and Celsus there is no reliable evidence that the
▪ Drinks blood of fallen sickly pope accepted the blood at all.
gladiators to gain strength - Most likely apocryphal (no basis); Anti-semitic
and vitality and to cure and Anti-Catholic
epilepsy;
▪ Taurobolium, the practice of 1615: Andreas Libavius
bathing in blood as it is - 1st person to advocate transfusion
cascaded from a sacrificial - Not known to have actually perform a
bull, was practiced transfusion
- ‘Leechdom’ practice - “Let there be a young man, robust, full of
o Painless procedure using leeches spirituous blood, and also an old man, thin,
o Injects anaesthetics substances— emaciated, his strength exhausted, hardly
explains the numbing feeling able to retain his soul. Let the performer of
o Injects anticoagulant the operation have two silver tubes fitting
▪ This will result to the difficulty into each other. Let him open the artery of
of ongoing bleeding since it the young man, and put it into one of the
will not clot tubes, fastening it in. Let him immediately
▪ Thus, potentially dangerous after open the artery of the old man, and
for patients that seeks put the female tube into it, and then the two
bleeding to stop tubes being joined together, the hot and
spirituous blood of the young man will pour
Early Blood Letting into the old one as it were from a fountain of
- Became so popular that the barbers life, and all of his weakness will be dispelled.”
became professional blood letters
- Their practice of hanging blood stained Transfusion Problems:
bandages outside the barbershop was the - Circulation
origin of the red and white stripped barber - Clotting / Coagulation
poles - Red Cell Metabolism
o Before, hanging them is used as
advertisement after an operation Circulation:
- Understanding the concept of circulation
There is power in the blood… was critical to developing the reality of
- The word ‘blood’ has been found 392 times blood transfusion
in the King James Bible: - Ancient Greeks:
o Genesis 4:10-11
1
LECTURE NOTES BY: JOASH-EVANGEL Z. CAMPOS, RMT, MLS (ASCPi)
o Blood was formed in the heart, then Xenotransfusion & Banning of Blood Transfusions
consumed as it flow out to the body - ‘Xenos’ = stranger, alien, foreign
in veins, while air was passed form - 1667: Jean-Baptiste Denys
the lungs to the body in arteries. o Performed the 1st fully documented
- Erasistratos ~ 270 BC: animal to human transfusion by
o Envisioned that the heart is a pump transfusing sheep blood to a boy
- Galen 131-201 AD: ▪ The rosetting of T cells by sheep
o Proved that arteries contain blood, erythrocytes is mediated through
but thought that blood was formed the interaction of the CD2
molecule on T cells with T11TS, a
in the liver, not suspecting that
molecule on sheep erythrocytes
arteries and veins are attached.
homologous to lymphocyte
- Andrea Cesalpino 1519-1603: function-associated antigen-3
o Used the term ‘circulation” (LFA-3, CD58)
o Believed that the veins and arteries o Denys and Emmerez performed
were connected by a fine vascular transfusion of lamb blood into the
network carotid artery of a young woman
- William Harvey 1616: ▪ the woman passed out black
o Explained the circulation of blood, urine following transfusion and
both pulmonary and systemic there was a finding indicative
circulation of a hemolytic transfusion
o Generally credited with the discovery reaction, but she still survived
in 1616 (published in 1628) of the o Denys’s fourth transfusion recipient,
circulation of blood as we know it suffering from luetic madness,
today* following a symptom-free first
*Blood is pumped from the left ventricle of the heart transfusion of calf blood, developed
through the aorta and arterial branches to the arterioles a hemolytic reaction upon his
and through capillaries, where it reaches an equilibrium second transfusion:
with the tissue fluid, and then drains through the venules ▪ “As soon as the blood began
into the veins and returns, via the venae cava, to the right to enter into his veins, he
atrium of the heart. felt ...heat along his arm,
and under his arm pits. His
Early infusion experiments: pulse rose presently, and
1658: William Boyle and Christopher Wren soon after we observed a
- performed a series of experiments injecting plentiful sweat over all his
various medicaments into the veins of dogs face. His pulse varied
utilizing a bladder with an attached quill extremely at this instant, and
and then observing the effects he complained of a great
- Infusion solutions included: pain in his kidneys, and that
o wine he was not well in the
o beer stomach, he
o opium awakened…He made a
o water great glass full of urine, of a
o nitric acid color as black, His madness
o sulfuric acid seemed improved, so
- Injected dyes into the blood vessels another transfusion was
supplying the brain in order to trace its undertaken which
vasculature (thus the Circle of Willis) unfortunately proved fatal.”
▪ This man’s wife charged him
Animal to Animal Transfusion with poisoning her husband.
- 1600: William Harvey (English physician) Denis was exonerated (and
o 1st known physician to describe the the wife was charged with
systemic circulation completely attempting to poison her
- This knowledge lead to direct blood husband!)
transfusions o This led to a 1678 prohibition by the
- 1665: Richard Lower (English physician) French Parliament of further
o 1st to successfully perform animal to transfusions.
animal blood transfusion by using o The British Royal Society (1668) and
dogs in his experiment the Vatican (1669) had also laid
o He kept dogs alive by connecting prohibitions against blood
the carotid artery of the donor dog transfusions.
to the jugular vein of the recipient o These prohibitions and the fear of
dog with a quill. adverse reactions led to a 150 year
o Dog 1 (carotid artery) to Dog 2 long near complete hiatus in
(Jugular Vein) transfusion work.
2
18th Century: - Milk was advocated as a potentially

- Transfusions were done only sporadically effective infusion, because it was thought
and were animal to human that the “white corpuscles.”
- Transfusion was generally thought of as cure - Two instances of successful transfusion, both
for mental aberration or as a youth potion administered during leg amputation, are
for the aged rather than as a treatment for documented from the Civil War.
blood loss.
- Reciprocal transfusions were suggested as a
Discovery of ABO Blood Types
cure for marital discord.
- 1901: Karl Landsteiner (Austrian biologist)
- Blood was thought to carry the
o Discovered the ABO blood group
characteristics of the donor to the recipient:
o Collected blood from his staff &
o Sheep’s blood would make a dog
mixed the different red cells and
grow wool, hooves, and horns;
serum
o Cat’s blood would make a girl feline,
o Was then able to demonstrate that
etc.
the serum of some people can
agglutinate the red cells of others
Human to Human Transfusion
o A major medical breakthrough
- 1818: James Blundell (English obstetrician)
o Packed Red Blood Cells of O+ is the
o Credited for performing the 1st
universal red cell donor & for
human to human blood transfusion
emergencies
and for rekindling the interest in
o 1902: Together with Adriano Sturli &
transfusion medicine
Alfred von Decastello discovered the
▪ Attentive human innovative
rarer AB Blood Type
approach; attempted
o 1930: Nobel Prize Laureate
human to human transfusion
of a man suffering from
Important Milestones:
gastric carcinoma
- 1907: Reuben Ottenberg (American
▪ Blundell’s transfusion devices
physician)
included the impellor which:
o Performs the 1st blood transfusion
a. Consisted of a cup,
using blood typing and cross-
tube and syringe
matching
b. A receptacle held
high above the
patient with an
attached tube
through which the
blood was injected
into the patient
o His experiments at the time were still
too primitive
o Thought that the exposure of air was
the reason for clotting
o He used a:
▪ Cannula or Y-shaped tube: to
avoid the air but still
frequently encountered
- 1937: “Blood Bank” coined
blood clots
o Dr. Bernard Fantus at Chicago’s
Cook Co. Hospital coins the term
19th Century:
‘blood bank’
- Transfusion became problematic because
- 1939-1940: Karl Landsteiner, Alexander
of complications of transfusion reaction
Wiener, Philip Levine & R.E. Stetson
- Panum and Landois showed that transfusing o Discovered the Rh blood group
blood with the same species are better and system
more efficacious than interspecies o Leads to dramatic decrease in the
transfusion. incidence of hemolytic disease of
- 2 observations of Landois in interspecies the newborn.
transfusions: o Over 250 different antigens
(1) Red blood cells were hemolyzed categorized into 23 major discrete
(2) White blood cells would cease their systems are now known.
amoeboid motion and die
- However, animal to human transfusions
were performed as late as 1890.
- Saline infusion was observed to be safer
than, frequently as effective as blood
transfusion.
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- 1939: Levine and Stetson o Recommended sodium bicarbonate

o described a severe reaction in a o Braxton Hicks unsuccessfully used
Type O woman given a transfusion sodium phosphate
of her husband’s Type O blood - Lewisohn (1914)
following a stillbirth. Her serum o Used citrate
agglutinated 80% of Type O blood. o Weil noted that citrated blood could
- 1941: Impact of War be stored in the refrigerator for
o The Red Cross begins National Blood several days
Donor Service to collect blood for o Lewisohn’s Method of Transfusion
the U.S. military Blood is collected in a citrated flask
o Soldiers injured during the Pearl and immediately transfused
Harbor attack are treated with - Kimpton-Brown (1918)
albumin for shock o Transfusion apparatus was
▪ During that time to be able to commonly used before citration.
separate components ▪ Consisted of a paraffin-
o Blood Center: collection, processing, coated gradient glass
etc. of blood cylinder with a horizontal side
o Blood Bank: stores blood tube for suction
Modern Blood Transfusion (3) Preservation
- 1914: - Rous and Turner
o Discovery of the chemical sodium o Furthering the work of Lewisohn and
citrate kept blood from clotting and Weil, they developed a solution of
allowed it to be stored for several salt, isocitrate and dextrose to both
days anticoagulated and preserve blood.
o Today, variations to this o This mixture made the blood
anticoagulant have been employed extremely dilute, so it had to be
for longer storage of blood removed prior to transfusion. (1:1
- 1950: solution:blood ratio)
o Disposable plastic blood bags o This method, with minor variations,
replaced glass bottles was used through most of World War
o Allowed convenient and efficient II.
blood transfusion as each blood bag - Loutit & Mollison (1943)
could be processed into several o Introduced ACD (Acid-Citrate-
components to maximize the benefit Dextrose) as a preservative
of a single donation o It was adopted by the Army in 1945.
o Glass bottles were used before and (1:4 solution:blood ratio)
was really inconvenient - 1957:
__________________________________________________ o ACD preservative was supplanted by
MAJOR INNOVATIONS IN THE 20th CENTURY: citrate-phosphate- dextrose (CPD)
➢ Compatibility ➢ Venous Access - 1965:
Testing ➢ Plastic Blood Bags o CPD with Adenine
➢ Anticoagulant ➢ Component - 1980:
Solutions Administration o CPD-A1
➢ Preservative ➢ Infectious Disease
Solutions Testing (4) Refrigeration
➢ Refrigeration ➢ High-Risk Donor - Effective preservation and refrigeration lead
➢ Blood Banks Screening to the ability to bank blood.
- 1960: Cryoprotective agents, such as
(1) Compatibility Testing glycerol, gain use in the 1960s, enabling
- Karl - Hektoen freezing of blood for long-term storage
Landsteiner - WWI
- Ottenberg & experiences (5) Blood Banks (1937)
Schultz - Coombs - During the Spanish Civil War, the Republican
Army banked 9000 liters of blood later
(2) Anticoagulation administered at casualty stations and base
- James Blundell hospitals.
- Bischoff (1835) - Bernard Fantus, at Chicago’s Cook County
o Proposed defibrination (whipping Hospital, established the first blood bank in
and twirling blood) the United Stated in 1937.
- Brown-Sequard (1850) - Blood banks now standard in communities
o Also experimented with defibrination and hospitals, with regional blood centers
o Was generally accomplished by collecting approximately 75% of the blood
whipping or twirling the blood, then supply for the United States.
removing the clot and transfusing - 13,588,000 units collected in the US in 1992.
the remaining fluid
- Neudorfer (1860) -
4
(6) Plastic Blood Bags

- Blood was collected into reusable glass
bottles in the first half of the twentieth
century. Whole blood was transfused.
Pyrogenic reactions from contamination due
to incomplete cleaning were frequent. Air
embolism was a common complication due
to the vacuum systems used on glass bottles.
- In 1949, trials of plastic bags were conducted
by the American Red Cross. Plastic bags
were disposable and, because of their
flexibility, facilitated the separation of blood
components and the advent of component
therapy.
- At least 17 different components are
available through a blood bank.
(7) Component Therapy

- Every unit of blood can treat more people.
- Revolutionized the treatment of
Haemophilia A.
CHANGES OVER TIME

- In 1943, Beeson described posttransfusion
hepatitis.
- The donor pool has changed from a
frequently paid group to a mandated
voluntary donation system.
- The worldwide pandemic of Human
Immunodeficiency Virus.
- Transition from Blood Banking to Transfusion
Medicine.
________________________________________________
Summary?
 The first attempts at blood transfusions were
primitive and cost a lot of lives.
 The discovery of blood types has made safe
blood transfusions possible.
 Through the help of blood banks and blood
centers, a single blood donation can be
maximized to benefit many.
 There’s no need for animal or human
sacrifice nor ritualistic dances to save a life.
 The magic of saving lives circulates in all of
our veins. The power to heal is only one
donation and one transfusion away.
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APPLICATIONS OF IMMUNOLOGY & SEROLOGY IN
BLOOD BANKING
B-cell
- Bursa of Fabricius
o Discovered by Hieronymus Fabricius
o Giving immunity
o Equivalent to the BM
T-Cell
- Only can recognize ‘self’ & ‘non-self’
- MHC (positive selection), Affinity of Tc to
MHC (negative selection)
Naïve/Virgin Cells (Th0, Tc0)

- Will get antigenic exposure in the peripheral
lymphoid organ
**Adaptive is more powerful due to specificity &

memory
**Anamnestic (Memory) Response: A rapid,
heightened secondary response against an antigen
upon reexposure as a result of circulating B-memory
cells, T4-memory cells, and T8-memory cells
**Douglas & Wright

- Tried to rationalize the difference between
the supporters of cellular and humoral
immunity
- Claimed that both functions were equally
important and interdependent
Complement System
- Support the action of the immune system
- Made out of proteins (under innate)
- Soluble; they need to be called & fixed to
activate
- All the numbers were based on their order of
discovery, not activation
- C3 is powerful due to more opsonization &
phagocytosis
Classical Pathway (specific)

o CRP + Ligand, Apoptotic cells, Ag-Ab
** Neutrophil → hrs (half-life: 6 hrs) o C1qrs (tulip-like structure complex)
** Major Basic Protein (MBP) o C4b2a (for boards), C4b2b
** NK: perforin + granzymes; cytotoxic for tumor (medschool)
cells. Viral infected cells, bacterial, fungal and o Amplification stage: Cleaving C3
parasitic infection o C5a: anaphylatoxin & chemotaxin
o Macrophage: long-lived compared
to neutrophils
o C8: the one that anchors (hole)
o C9: poly-C9 (walls), C9n**
** ‘n’ means infinite number
6
“All immunogens
Lectin Pathway (non- are antigens, but
specific) not all antigens are
o Lectin: protein immunogens.”
capable of attaching
to cell surfaces
o Mannose: found in microorganisms • Antigen: capable of reacting with an
MASP-1, 2, 3* (*stevens) = C1qC1rC1s antibody
• Immunogen: initiates an immune response
Alternative (Properdin) Pathway • Hapten: small molecules that do not elicit an
o Pathogen Associated Molecular immune response on their own; needs a
Patterns carrier
▪ Tells the immune system that
they’re pathogens Antigen Properties that Influence Immune Response:
▪ C3b recognized them 1. Foreignness
o Pattern Recognition Receptors (PRRs) 2. Size
▪ Hydrolzed by H2O (plasma) 3. Complexity
o Bypass several (goes directly to C3) 4. Accessibility (Epitope / antigenic
determinants)
5. Digestibility (integrity of the antigen)
6. Stability
7. Conformation (linear & discontinuous)
8. Chemical composition
o Properdin: stabilizes C5 convertase
** HLA-DMA and -DMB genes are both required

for MHC-II (CD4+ Tc)
**IL-4: Bc growth factor (paracrine signalling)
**MHC-1 is a distress signal to all nucleated cells
except RBCs
RBC Antigen Compositions:

- RBC antigens are very diverse in structure
and composition and may be:
o Glycoprotein (Rh, M, N)
o Glycolipid (ABH, Lewis, Ii, P)
o Protein (HLA)
**Endonucleasae helps to lyse cells; **Allo = other individual but same species
produced/released by bacteria
**If you feel hot or not feeling well within the 15 Antibodies at a glance:
minutes of transfusion, stop the transfusion • Valency: number of antigen binding sites
immediately • Epitope: antigenic determinant
• Paratope (antigenic binding site)
7
o Clusters of complementary- Immunoglobulin Gamma (IgG)
determining regions (CDRs) in - Warm-reacting antibody
hypervariable regions - 37 degrees Celsius
- Can cross placenta
o IgG1 (+), IgG2 (±)
o IgG3 (+), IgG4 (+)
- Can fix complement though not as efficient
as IgM (needs 2 CH2):
o IgG1 (++), IgG2 (+)
o IgG3 (+++), IgG4 (0)
- Most of the clinically significant Abs (affinity
to antigen)
- Can cause anaemia and transfusion
reactions
Immunoglobulin Mu (IgM)
- Cold reacting antibody
- 4 degrees Celsius
- Room temperature (‘RT Antibody’)
- ABO blood group system
- Naturally occurring antibody
- Primary testing problem:
o IgM antibodies interfere with
**Secretory protein/piece – to protect once in the detection of clinically significant IgG
secretion antibodies by masking their reactivity
**Hypervariable Region – contribute to the - Remedy to take out IgM: B2-
specificity of each antibody mercaptoethanol (2-ME) or dithioreotol
**Variable region – A.A.s here change accord. to (DDT)
the specificity of the Ag - Take out IgG: ZZAP (thiol-reagent +
**Heavy chain (constant region) is where you can proteolytic enzyme
determine what Ig it is
Characteristics of Blood Group Antibodies:
1. Polyclonal vs Monoclonal Antibodies
• Antisera (reagent antibodies)
• Polyclonal/Serum Antibodies
o Produced in response to a single
antigen with more than 1 epitope
• Monoclonal Antibody
o Antibody formed by a single type of
B cell, clonally expanded, against an
antigen of a single type of epitope
**About 600 molecules (probability for 2 IgGs to

activate complement)
**Fc portion – crystallizable due to pepsin
**Interchain: join 2 chains together; Intrachain: sth.
that’s inside the chain
**Affinity – predominant ang IgG; Avidity –
predominant ang IgM
**Papain – cleaved above the hinge region
**Pepsin – cleaved below the hinge region
__________________________________________________
_________
Hybridoma Technology
- Produced by using polyethylene glycol
**Atopic individuals have the tendency to be (PEG) to fuse cells
hyperallergic - Myeloma Cells: immortal growth properties
**IgD = B-cell surface (mature); associated with the - B-Cells: to contribute the genetic
development information for synthesis of specific antibody
- Selected using Hypoxanthine, Aminopterin &
Thymidine (HAT) Medium
8
2. Naturally occurring vs Immune antibodies
• Naturally Occurring Antibodies (NOA)
o a.k.a Isoagglutinins Characteristics of Antigen-Antibody Reactions:
o Found in the serum of patients who 1. Intermolecular binding forces
have never been exposed to that a. Hydrogen
antigen bonds
o Mostly IgM b. Electrostatic
• Immune Antibodies (IA) forces
o Found in the serum of patients who c. Van der waals
have been exposed to antigen forces
through prior transfusion or d. Hydrophobic
pregnancy bonds
o Mostly IgG e. Hydrophilic
bonds
MOST COMMON MOST COMMON 2. Antibody properties
NOA IA a. Affinity vs Avidity
• ABH • Rh b. Specific reaction
• Hh • Kell c. Cross-reaction
• Ii • Duffy d. No reaction
• MN • Kidd 3. Host factors
• Lewis • Ss - Status, age, race, etc.
• P 4. Tolerance
**Agglutinin (Ab); Agglutinogen (Ag)
Factors that influence agglutination reactions:
1. Charge
Unexpected antibodies?
2. Centrifugation
- Decreases reaction time by increasing
- These may be due to previous transfusion, gravitational forces and bringing reactants
pregnancy, etc. closer together
- Sensitized RBCs overcome their natural
3. Alloantibodies or Autoantibodies repulsive effect (zeta potential) for each
2 unexpected red cell antibodies: other and agglutinate more efficiently
• Alloantibodies
o Produced after exposure to
genetically different, or non-self-
antigens such as after transfusion
(RBCs, WBCs, or even platelets not
present in patient)
• Autoantibodies 3. Antigen-Antibody ratio
o Produced in response to self- - Prozone: excess of unbound antibodies
antigens causing false negative reaction
o Can react at both warm & cold - Postzone Effect: surplus of antigen causing
temperatures false negative reaction
**Antibody Panel: extended ver. of an antibody - Zone of Equivalence: number of binding
screen; has 10 panel cells sites of multivalent antigen and antibody
are approximately equal
9
Immediate Spin Antiglobulin Phase /

Phase (IgM) 37˚ (IgG)
A, B, H Ss
I, i Kell (Kk, Jsa, Jsb, Kpa,
M, N Kpb)
Lewis (Lea, Leb) Rh (DCEce)
Lutheran (Lua) Lutheran (Lub)
P Duffy (Fya, Fyb)
Kidd (Jka, Jkb)
7. Enhancement media
- Sialic acid on RBC
**Prozone – do serial dilution (remedy); heavy membranes causes
infections RBCs to repel each
**Postzone – add more serum from (remedy); early other since most acids
stages of infection have a negative
**Grading agglutination reactions: charge and therefore
- To standardize element of subjectivity creates this large
among Blood Bank personnel performing ‘zone’ of negative
the testing charge around the cell
- May be performed in test tubes, - Zeta potential:
microplate wells, microtubes filled expression of the
with gel particles difference in the
- Conventional grading for the tube electrostatic charges at the RBC surface
system uses a 0 to 4+ scale and the surrounding cations (Abs)
- Reduces the zeta potential of RBC
membranes:
a. Saline Media
b. Protein Media
c. Low Ionic Strength Solution (LISS) Media
d. Proteolytic Enzymes
8. Protein media
• Colloidal substances
- Type of clear solution that contains
4. Effect of pH particles permanently suspended in
- pH (power/potential of hydrogen)—figure solutions
expressing the alkalinity or acidity of a o Can be charged/neutral
solution Examples
- Ideal pH of a test system: pH 6.5-7.5 ▪ 22% ▪ Polyvinylpyrolli
- Normal blood pH is tightly regulated at 7.35- Albumin done (PVP)
7.45 ▪ Polyethyl ▪ Protamine
- Except these antibodies which react best at ene
pH<6.5: glycol
o Anti-M (PEG)
o Pr(Sp1) ▪ Polybrene
- Acidification of test serum may aid in • Crystalloids
distinguishing anti-M and anti- Pr(Sp1) - Small, highly soluble molecules that
antibodies from others are easily dialyzed
5. Temperature - Example: Glucose
• Immediate spin (IS) phase:
o Ambient room temperature 9. Low ionic strength solution (LISS) /Low Salt
o <22 degrees Celsius Media
o IgM antibodies - Generally contain 0.2% sodium chloride
• 37 degrees incubation phase: - Increase rate of antibody uptake during
o IgM antibodies sensitization
• Antihuman globulin (AHG) phase: - Compared with protein potentiators /
o IgG antibodies media, LISS decreases incubation time
(from 30-60 minutes to 5-10 minutes)
6. Immunoglobulin type - May result in false-positive reactions and
- Differences in the nature of reactivity may require testing to be repeated with
between IgM and IgG antibodies require albumin
different serologic systems to optimally
detect both classes of clinically significant
antibodies
10
10. Proteolytic enzymes - Structure and location of antigen present on
• Enzymes: the red cells is what make each blood
- protein molecules that function by group system unique
altering reaction conditions and bringing
about changes in other molecules
without being changed themselves
• Substrate:
- molecule that enzymes react with
• Proteolytic enzymes:
- target protein molecules
• Enzymes release sialic acid from RBC
membrane thus decreasing and negative
charges
Enhanced Decreases
Antibody Antibody
Reactivity Reactivity
Rh Fya
Kidd Fyb
P1 M
I N
Lewis S Terminology:
Gene
Summary: - Basic unit of inheritance that codes a
- That having a strong foundation in particular protein
immunology and serology can help us - Usually written in italics
understand the principles in blood
banking and transfusion medicine Chromosomes
- That antibodies have different - Double strands of DNA where genetic
characteristics thus it is very crucial for information is carried on
blood bank personnel to detect and
investigate them properly Allosome
- That antigens have different properties - Sex chromosome
which influence antigen-antibody
reactions Autosomes
- That technology has allowed more - Any other chromosome that is not an
efficient work in the blood bank that aid allosome
in testing and faster response to patient
care Meiosis
BASIC GENETIC PRINCIPLES IN BLOOD BANKING - Cell division in gametes
Genetics Phenotype
- Defined as the study of inheritance or the - Physical expression of inherited traits
transmission of characteristics from parents - Determined through hemagglutination
to offspring
- Based on the Genotype
o Biochemical structure of chromatin, - Actual genes inherited from each parent
which includes nucleic acids - Determined through family studies /
o Various enzymes that assisting molecular typing
genetic processes such as
replication and division Early Genetics:
o Structural proteins that constitute the Carolus Linnaeus
genetic material - Swedish biologist who started the first
classification system of living things in the
17th century
- Used ‘species’ as the principal unit of
definition
Charles Darwin
- “On the Origin of Species”
Blood Group Systems - Ambition was to understand the diversity of
- Groups of antigens on the red cell life
membrane that share related serologic - Natural Selection
properties and genetic patterns of o Differential survival of individuals due
inheritance to differences in phenotypes
11
Gregor Mendel’s Law of Inheritance Suppressor Gene
- Genes that act to inhibit the expression of
another gene to produce a null expression /
null phenotype
- Rare occurrence
- Examples:
o In(Jk) gene affects the Kidd blood
group system expression, causing the
Jk(a-b-) phenotype
o In(Lu) gene suppresses the Lutheran
blood group system, resulting in the
Inheritance Patterns: Lu(a-b-) phenotype
Dominant
- Gene product expressed over another gene Punnet Square
- Written in capital letters (i.e. RR) - Used to display the frequencies of different
genotypes and phenotypes among the
Recessive offspring of a cross
- Trait expressed when inherited from both - Illustrates the probabilities of phenotypes
parents only when the dominant gene is from known or inferred genotypes
absent
- Written in small letters (i.e. rr)
Codominant
- Equal expression of both inherited alleles
from parents found on autosomes
- Pattern of inheritance in most blood group Silent Genes (Amorphs)
antigens - Do not produce a detectable antigen
- Written in capital letters (i.e. AB) product
o Phenotype is called Null Types
Where are genes located? - Amorphic genes
must be inherited
from both parents
(homozygous) to
product a null
Allele phenotype
- Different forms of a gene - Examples:
o O
Antithetical Gene o Rhnull
- Meaning ‘opposite’ o Lu(a-
- Antigens produced by alleles b-)
- Example:
o Kpa & Kpb (alleles) Genetic Interaction
o Kpa is antithetical to Kpb - Interaction of genes
depending on how they are inherited:
Dosage: o Cis = same chromosome
Homozygous o Trans = opposite chromosome
- Individual whose genotype is made up of - trans genes can weaken the expression of
identical genes, such as AA, BB, or OO the gene opposite
- Individual called a homozygote - Position Effect
- “double dose”
Heterozygous
- Individual who has inherited different alleles
from each parent, such as AO, AB or BO
- Individual called a heterozygote
- “single dose”
Chromosomal Assignment of Blood Group Systems
- Most of the blood group-associated genes
are on autosomes, except Xg blood group
where the gene is found on the X
chromosome
12
BLOOD GROUP CHROMOSOME Polymorphism
SYSTEM - Refers to having 2 or more alleles (versions)
Rh 1 at a given locus, as with the ABO blood
Duffy 1 group system
MNS 4 - Rh Blood Group System is highly
Chido/Rodgers 6 polymorphic than ABO due to the greater
Kell 7 number of alleles
ABO 9 - Disadvantage:
Kidd 18 o HLA polymorphism contributes to
Lewis 19 challenge of finding suitable donors
- Advantage:
o Can be used in genetic studies or
Mendelian Principles / Laws
Independent segregation relationship testing
- Passing of one gene from each parent to
offspring
- Refers to the transmission of a trait in a
predictable fashion from one generation to
the next
Independent assortment
- Random behaviour of genes on separate
chromosomes during meiosis that results in a
mixture of genetic material in the offspring Relationship Testing:
Direct Exclusion
- Child has inherited a genetic marker that’s
not found in the mother or alleged father
Obligatory Gene
- Genetic marker passed by the father to
establish probability of paternity
Exception to the Law of Independent Assortment
Linkage & Haplotypes:

Linked Genes
- Genes that are very close
together on a Indirect Exclusion
chromosome - Child lacks the genetic marker that the
father should have given to all of his
Haplotype offspring
- Set of linked genes
inherited together
because of their close proximity on a
chromosome
Pedigree Analysis
Crossing Over: - The study of an inherited trait in a group of
- Exchange of genetic material during meiosis related individuals to determine the pattern
after the chromosome pairs have replicated and characteristics of the trait, including its:
- Occurs when two genes on the same o Mode of inheritance
chromosome recombine o Age of onset
- Rare phenomenon but can cause the o Phenotypic variability
predicted inheritance patterns to be - Males are always represented by squares
changed - Females are always represented by circles
- Mating
o Line joining a male & female
- Stillbirth / Abortion
o Small black circle
- Deceased family member/s (line across)
- Propositus
o Indicated by an arrow pointing to it
13
Applications of Molecular Testing in BB:
14
ANTIGLOBULIN TESTING
Categories of Reagents in BB Lab: filter through a gel matrix in the gel microcolumn
• Reagent red cells method (B), or the adherence of red blood cells
• Antisera onto the surface of a well in solid phase methods
• Antiglobulin reagents (C).
• Potentiators
US Food & Drug Administration (FDA) - Discovery of the test:

- Provides regulations for the blood bank o 1908: Described by Moreschi,
industry (includes licensing of blood bank ▪ Used rabbits and goats in his
reagents) experiment
- Publishes the Code of Federal Regulations o Coombs procedure involved the
o Outlines the FDA criteria for the injection of human serum into rabbits
licensure of blood bank reagents to produce antihuman serum
▪ Used to detect any IgG antibody
Specificity that has attached to an RBC in
- Reflects the unique recognition of the vivo or in vitro
antigenic determinant and its - Principle:
corresponding antibody molecule o AHGs obtained from
- E.g. Anti-D reagents only react with D immunized nonhuman
antigens in sample species bind to human
globulins (IgG or
Potency Complement) attached to
- Addresses the strength of the antigen- antigens on red blood cells that
antibody reaction happen in vivo or in vitro
- E.g. commercial antisera should agglutinate - Purpose:
strongly (3+ or 4+) o Detect RBCs sensitized with IgG
alloantibodies, IgG autoantibodies,
**Reagents should not be used beyond expiration & complement components
date (except for rare antisera and red cells as long - Sensitization Phases:
as they pass QC results) o In vivo sensitization
▪ Red blood cells have been
Major Types of Blood Group Antibodies: attached with antibodies directly
• IgM inside the body
o Pentameric structure, easily agglutinates ▪ Technique: Direct Antiglobulin
RBCs Test
o Complete antibodies ▪ One-stage procedure
• IgG o In vitro sensitization
o Monomeric structure ▪ Red blood cells have been
o Too small to directly agglutinate attached wit antibodies as
sensitized RBCs observed in a laboratory
o Nonagglutinating/incomplete antibodies ▪ Technique: Indirect Antiglobulin
Test
Antihuman Globulin Test / Antiglobulin Test / ▪ Two-stage procedure
Coomb’s Test
- 1945: First introduced by Cambridge AGT Reagents:
immunologists, Robin Coombs, Arthur Polyspecific AHG
Mourant, & Rob Race - Contain antibody to human IgG & c3D
- Great value in investigating Rh HDFN cases - May also contain Anti-C3b, Anti-C4b, & Anti-
and later on discovered to have versatility in C4d
detecting other IgG blood group antibodies - C3d & C4d are usually the only fragments of
complement that remain attached to red
Negative and positive direct cells due to spontaneous denaturation
antiglobulin test (DAT) - Commercially prepare polyspecific AHG
reactions. The DAT detects may contain little activity against IgA and/or
immunoglobulin and/or IgM heavy chains
complement on the surface - However, may also contain antibody
of red blood cells, which against kappa or lambda light chain
agglutinate with the anyway and thus can react with all
addition of antihuman immunoglobulins
globulin. The agglutination is - Prepared using polyclonal or monoclonal
detected visually as clumps technology
of red blood cells by the
conventional test tube
method (A), by the failure to
15
Monospecific AHG
- Contain only 1 antibody specificity: either
Anti-IgG (specific for Fc fragment of IgG) or
Anti-C3b-C3d (anticomplement blend)
- Prepared using polyclonal or monoclonal
technology
Preparation of AHG:
Polyclonal AHG Production
- Produces a mixture of antibodies from
different plasma cell clones
- Usually prepared by injecting separate
colonies of rabbits
o Hyperimmunized to produce high-
titer, high-avidity IgG antibodies
- Titration done to avoid prozone (false
negative test results)
- Determining the level of Anti-3d is critical in
keeping false-positive tests to a minimum
- When large volumes of reagent are
required, sheep or goats may be used by
the manufacturers
Monoclonal AHG Production

- Derived from one clone of animal plasma
cells that recognize a single epitope
producing high-titer antibodies with well-
defined specificities to IgG & to the
fragments of C3
- Produced y hybridoma technology using
mice Anticomplement Activity
- Devised by Kholer & Milstein - Some antibodies are capable of ‘fixing’
- Resulting monoclonal IgG (mixture of IgG1 & complement components to the RBC
IgG3) reacts with CH3 region of gamma membrane after complexing of the
heavy chain of human IgG subclasses 1, 2, antibody with its corresponding antigen
&3 (end goal: cytolysis)
- Further degradation of membrane-bound
C3b & C4b occurs by removing C3c & C4c
to leave C3d & C4d firmly attached to RBC
membrane
- To use polyspecific or monospecific AHG?
o Polyspecific AHG Advantage: can
detect RBCs sensitized by IgG or
complement
o Polyspecific AHG Disadvantage:
false positives to non-clinically
significant IgG antibodies
o Many blood banks prefer to use
polyspecific monoclonal AHG for
routine transfusion testing
- Verdict: deciding to use which AHG reagent
is the prerogative of each individual blood
bank
ANTIBODIES CAPABLE OF BINDING COMPLEMENT

MOST SOME RARE
ABO, Le , Le , S, Xg , LKE, Lan
a b a S, P1, Lua, Lub,
Jk , Jk , Sc1,
a b Kell, Fya, Fyb,
Co3, Ge2, Cob
Ge3, Ii, P, P ,
k
Vel
16
DIRECT ANTIGLOBULIN TEST (DAT) PANEL

- Purpose: detects in vivo sensitization
- Applications:
o Hemolytic Disease of the Fetus &
Newborn (HDFN)
o Hemolytic Transfusion Reaction (HTR)
o Autoimmune & Drug-induced
Hemolytic Anemia (AIHA/DIHA)
- Not a required test in pretransfusion testing
protocols
- One drop of a 3-5% washed red blood cell
suspension from patient + polyspecific (Anti-
IgG, Anti-C3d) AHG
DAT Panel: Patterns of Reactivity in AIHA

Anti-IgG Anti-C3d Type of AIHA
+ + WAIHA
+ - WAIHA
- + CAS; PCH;
WAIHA
+ + Mixed-type
AIHA (cold &
warm)
Confirm Negative Results:

IgG Sensitized Red Cells / Coombs Control /
Coombs Check Cells/ Check Cells
- Consists of red cells that have been
prepared with IgG Abs attached & used as
a commercial control system
- Confirms true negative DAT & IAT results
- The check cells should react with the free
AHG reagent and show agglutination (+)
- What if the result is still negative when
INDIRECT ANTIGLOBULIN TEST (IAT) PANEL adding check cells? Potential reasons for
- Purpose: detects in vitro sensitization false (-):
- Application: o Failure to add AHG reagent to the
o Detection of incomplete test
(nonagglutinating) antibodies to o Failure for AHG reagent to react
potential donor RBCs (compatibility (expired)
testing) or to screening cells o Failure to wash red cells adequately
(antibody screen) in serum
o Determination of RBC phenotype
using a known antisera (E.g. Kell
typing, weak D testing)
o Titration of incomplete antibodies
APPLICATION TESTS IN VITRO SENSITIZATION

Antibody Compatibility Recipient antibody
detection testing reacting with donor Factors affecting AGT:
cells (from blood bag - The number of IgG molecules that sensitize
component) an RBC & the rate at which sensitization
Antibody Antibody reacting with occurs can be influenced by several factors,
screening screening cells including:
Antibody Antibody Antibody reacting with
identification panel panel cells 1. Ratio of serum to cells
Antibody Rh antibody Antibody & selected - Increases the sensitivity of the test system
titration titer Rh cells
- Generally serum to cell ratio is 40:1
RBC phenotype Weak Specific antisera +
- Achieved by using 2 drops serum + 1 drop
antigen RBCs to detect antigen
detection 5% v/v cell suspension
17
2. Reaction medium - All negative antiglobulin test reactions must

- Albumin be checked by the addition of IgG-
o Macromolecules allow antibody- sensitized cellls
coated cells to come into contact
with each other so aggregations can
occur
o no advantage over LISS thus rarely
used as an IAT media for routine tests
- Low Ionic Strength Solution (LISS)
o Enhance antibody uptake and allow
incubation times to be decreased
(10-15 minutes) by reducing zeta
potential around RBC
- Polyethylene Glycol
o Water soluble linear polymer that
removes H2O thereby concentrating
Ab
3. Temperature
- Rate of reaction for majority of IgG: 37%
- Usual incubation temperature for IAT
- Also the optimum temperature for
complement activation
4. Incubation time
- For cells suspended in saline: 30-60 (120) min.
o Majority of clinically significant
antibodies can be detected after 30
minutes of incubation
o Extended incubation times not
usually necessary
- If LISS / PEG is used: 10-15 minutes
o Essential that tubes be incubated at
37° with this shortened amount of
time
o Extended incubation time (40
minutes) for LISS has shown antibody
5. Washing of RBCs
- Saline wash a minimum of 3x before adding
AHG reagent
- Inadequate washing may result in false
negative
- Cell pellet should be completely
resuspended before adding the next saline
wash Modified / Automated AGT Techniques:
- All saline wash be discarded after final wash → Low Ionic Polybrene Technique
6. Saline for washing - A potent rouleaux-forming agent that allows
- Saline should be fresh or buffered to a pH of sensitized cells to approach each other and
7.2-7.4 permit cross-linking by the attached
- Don’t ignore expiration dates as decrease in antibody
pH can happen when saline is stored for - Must use monospecific anti-IgG reagent if
long periods of time in plastic containers this technique is employed
- Bacterial contamination can also contribute - Disadvantage: low sensitivity detection of
to false-positive results anti-Jka and anti-Jkb
7. Addition of AHG → Enzyme-Linked Antiglobulin Test
- Added immediately after washing to - Amount of color produced is proportional to
minimize chance of elution the amount of antibody present
- Adding 2 volumes of AHG may overcome - Optical density is measured at 405 nm
washing problems - Uses a chromogen (spectrophotometer)
8. Centrifugation for reading
Sources of Error:
- EDTA blood samples are best for DAT to
avoid in vitro complement attachment
associated with refrigerated clotted
specimens
18
Antibody Potentiators and Lectins:

Antibody Potentiators (Enhancement Media)
- Commercially available reagents that
enhance the detection of IgG antibodies by
increasing their reactivity:
o May enhance antibody uptake
(sensitization / first phase of
agglutination)
o Promote direct agglutination
(lattice formation / second phase of
agglutination)
→ Solid Phase Technology

- Direct Test: antibody is attached to
microplate well and RBCs are added
- Indirect Test: known RBCs are bound to a Other Common Lectins:
well that has been treated with ➢ Anguilla Anguilla (eel): Anti-H Lectin
Glutaraldehyde or Poly L-lysine and ➢ Helix pomatia (snail): Anti-A Lectin
antibody is added ➢ Vicia graminea (plant): Anti-N Lectin
- Types of Wells: U-shaped & V-shaped ➢ Iberis amara (plant): Anti-M Lectin
bottoms
- Test serum is added to the RBC-coated
wells:
o (+) = bottom of the well will be
covered with suspension
o (-) = RBCs will settle to the bottom of
well
→ Gel Test
- Developed by Yves Lapierre (1988), it is a
chamber filled with dextran polyacrylamide
gel
- After centrifugation, free unagglutinated
RBCs form pellets at the bottom while
agglutinated cells are trapped in the tube
for hours
19
LECTURE NOTES BY: JOASH-EVANGEL Z. CAMPOS, RMT, MLS (ASCPI)
POTENTIATOR DESCRIPTION ADVANTAGE DISADVANTAGE

Low Ionic Strength Solution (LISS) → Principle described by Low & Reduced Enhances cold
→ Contains: Messeter (1974) incubation autoantibodies
o Sodium chloride → Na+ and Cl- ions in physiologic time (10-15
o Glycine saline cluster around Ag-Ab minutes)
o Salt-poor albumin complex hindering reaction
(approx. 0.03M) → LISS lowers the ionic strength of
→ Compared with saline (approx. the medium increasing uptake
0.17M) of Ag-Ab reaction
Polyethylene Glycol (PEG) → Water soluble polymer that Increases Enhances
→ Use only in IAT removes water in the test sensitivity by warm
→ Use only monospecific anti-IgG environment allowing greater detecting Abs autoantibodies
AHG reagent collision between Ag-Ab not found in
reactions LISS or BSA
Enzymes: → Proteolytic enzymes from plants Help solve the Enhances cold
• Ficin (figs) that break down protein identity of & warm
• Papain (papaya) molecules unexpected autoantibodies
• Bromelin (pineapple) → Possess the property to modify antibodies
Increases, decreases or inhibits Ab red cell membranes by Should be used
reactions removing negatively charged as a tool of
molecules and denaturing investigating
certain antigenic determinants discrepancies
not as a sole
method of ID
Bovine Serum Albumin → Mechanism of action: Enhances Reduces
→ Prepared from bovine serum or influences the second stage sensitivity of repulsion
plasma commercially available (lattic formation) of IAT. Does not between cells
at 22 or 30% concentration agglutination by allowing enhance but does not
antibody-sensitized cells to warm shorten
come closer to one another autoantibodies incubation time
20
ABO BLOOD GROUP SYSTEM (ISBT NO. 001)
ABO Blood System

- Individuals possess antibodies against
antigens they lack
- ABO antibodies are ‘naturally occurring’
Tube method can do incubation, centrifugation,
and are initiated at birth, but are too low for etc.
detection until individual is 3-6 months of
age Formation of A, B, & H: Red Cell Antigens
- Antibody production peaks at 5 & 10 years - ABH red cell antigens
of age then declines later result from the interaction
- 1901: Karl Landsteiner (ABO) of genes at 3 separate
- 1902: Adriano Sturli & Alfred von Decastello loci (ABO, Hh, & Se)
(AB) - Genes code for specific
glycosyltransferases
General Characteristics - A, B, & H antigens can be
- ABO antibodies are IgM in nature glycolipids, glycoproteins,
- ABO antibodies react preferentially at room or glycosphingolipids and
temperature (20-24°C) or below are formed from the same basic precursor
- Pentametric structure allows efficient material (paragloboside or glycan)
activation of complement at 37°C **MHC (Chromosome 6p); ABO (Chromosome 9q)
- Serum from Group O individuals contain not **They code for enzymes, which are catalytic
only Anti-A & Anti-B but also Anti-A, B reactive.
- Anti-A, B is not a combination of Anti-A & - H gene must be
Anti-B but a separate ‘cross-reacting’ inherited to form ABO
antibody of IgG nature antigens on the RBCs
- Since IgG can cross the placenta, knowing - ABO antigens can be
the amount of Anti-A, B antibodies in a found on the surface
woman’s serum or baby’s cord blood can of RBCs as well as in
sometimes allow prediction or diagnosis of secretions
HDFN - Se gene must be
- Anti-A, B antisera is used in the lab to inherited to form the
confirm group O units, not in routine testing ABO antigens in
secretions
ABO Grouping
o H & Se genes are closely linked &
Forward Typing / RBC Typing (Front Type) located on chromosome 19
- Defined as using known sources of o ABO genes are found on
commercial antisera (Anti-A, Anti-B) to chromosome 9
detect antigens on an individual or patient’s - The H (HH/Hh) gene codes for the L-
RBCs fucosyltransferase
o Enzyme attaches a fucose sugar to
Reverse Typing / Serum Typing (Back Type) the paragloboside structure forming
- Defined as detecting ABO antibodies in the the H antigen
patient’s serum by using known reagent - H antigen is the precursor structure on which
RBCs, namely A & B cells A & B Ags are made
- Paragloboside Chains:
o Type 1 & 3 associated with body
secretions
21
o Type 2 & 4 associated with the red - D-galactose confers group B specificity
cell membrane (610,000-830,000 B antigen sites) on an adult
- Most abundant types of precursor structures B RBC
on RBCs:
o Type 1 Chain: precursor structure in Formation of the AB Antigen
secretions - When both A & B genes are inherited, the B
o Type 2 Chain: precursor structure on enzyme (a-3-D-galactosyltransferase)
erythrocytes competes more efficiently for the H antigen
**Fucose: Immunodominant sugar especially for ‘H’ > A enzyme (a-3-N-
specificity acetylgalactosaminyltransferase)
- Ave. no. of A antigens on adult AB cell:
600,000 sites
- Ave. no. of B
antigens on adult
AB cell: 720,000
sites
Formation of the O Antigen

- Blood group O
individuals inherit at
least 1 H gene
(genotype HH/Hh)
Types of Precursor Chains: & 2 O genes (OO)
Type 1 Precursor Chain o O gene is an
- Refers to beta 1 → 3 amorph
linkage between - O gene does not
galactose & N- elicit production of a
acetylglucosamine glycosyltransferase
therefore, H antigens
Type 2 Precursor Chain remain unmodified
- Refers to beta 1 → 4 - H gene elicits the
linkage between production of an
galactose & N- enzyme, a-2-L-
acetylglucosamine fucosyltransferase,
which transfers the
• Expression of A & B antigens on RBCs fully sugar L-fructose to
develop by 2-4 years of age and remain an oligosaccharide
constant chain on the terminal
• RBCs of newborns estimated to carry about galactose of type 2
25-50% of the number of antigenic sites found chains
on the adult RBC - L-fructose: sugar
responsible for the H
Formation of the A Antigen specificity
A gene (AA/AO):
- Codes for the production of a-3-N- Formation of A, B, & H:
acetylgalactosaminyltransferase which Soluble Antigens
transfers an N-acetylgalactosamine - ABH-soluble antigens
(GalNac) sugar to the H antigen structure (substances) can be
- Tends to elicit higher concentrations of found in all body
transferase than B gene leading to secretions and are
conversion of practically all H antigens (as made up of
many as 810,000-1,170,000 A antigen sites glycoproteins
exist) - Their presence is
dependent on the
Formation of the B Antigen inheritance of the SeSe
B gene (BB/BO): or Sese (secretor gene)
- Codes for the production of a-3-D- which codes for a-2-L-
galactosyltransferase which transfers an D- fucosyltransferase
galactose (Gal) sugar to the H substance on
a type 2 precursor chain
22
o Enzyme modifies the type 1 precursor o Ulex eurpaeus

substance to form the H substance (anti-H):
which determines a secretor agglutinates O
o People who inherit the sese cells & other
genotype are nonsecretors ABO blood
groups
depending on
the amount of H
antigen
available
H Antigen
• Anti-H
- A naturally occurring
IgM cold agglutinin occasionally found in
the serum of A1 & A1B individuals due to
well-hidden H antigen on their RBCs
- Insignificant Ab in terms of transfusion
because of no reactivity at body
temperature (37°C)
- Anti-H lectin reacts weakly with the RBCs of
A1B individuals
ABO Subgroups
- ABO subgroups represent phenotypes that
show weaker variable serologic reactivity
with the commonly used human antisera
anti-A, anti-B & anti-A, B reagents:
o A Subgroups
o Weak A Subgroups
o Weak B Subgroups
A Subgroups
- 2 different A antigens described by Emil von
Dungern (1911) based on serologic
reactions
- More common than B subgroups
- A1: cells of approximately 80% of group
A/AB
- A2 / weaker A subgroups: remaining 20%
- Production of antigens is a result of the
inheritance of either A1 gene (more potent)
or A2 gene that codes for A transferase
- Immunodominant sugar: N-
Formation of Anti-A1
- Identification of 4 different forms/chains of H
antigens which corresponds to precursor
structures on which A enzyme can act to
acetylgalactosamine convert to A antigen:
o Unbranched straight straight chains
Characteristics of A1 & A2 Phenotypes: (H1 & H2): converted by A1 & A2
- About 1-8% of A2 & 22-35% A2B individuals (less efficiently) into Aa & Ab antigens
produce anti-A1 (IgM) in their serum o Complex branched chains (H3 & H4):
- Lectins used in BB: converted by A1 & A2 (very poorly)
o Dolichos biflorus (anti-A1 lectin): into Ac & Ad antigens
agglutinates A1B - A2 individuals with more unconverted
o Bandeiraea simplicifolia (anti-B complex branched H chains develop anti-
lectin): agglutinates B cells A1
23
- Infants appear as A2 at birth due to

deficiency of H3 & H4. Later developing into
A1 individuals in a few months.
Gradient of the subgroups of A: # of A antigen sites

per red cell
A1 (most A Ags) → A2, A3 → Ax → Ael (fewest A Ags)
Weak B Subgroups
- Occur less frequently than weak A
subgroups
- Criteria used for differentiation of weak B
phenotypes:
o Strength & type of agglutination with
anti-B, anti-A, B & anti-H
o Presence or absence of ABO
isoagglutinins in the serum
o Adsorption-elution studies with anti-B
o Presence of B substance in saliva
o Molecular testing
The Inheritance of the Rare h Allele:

Subgroups Weaker than A2
Bombay Phenotype (Oh/Hnull)
- Make up 1% of discrepancies encountered
- First reported by Dr.
in the lab and therefore mainly of academic
Bhende (1952) in
interest
Bombay, India
- Characteristics:
- Represents an
o Decreased number of A antigen sites
inheritance of double
per RBC (resulting in week or no
dose h gene (hh)
agglutination with human polyclonal
- As a result, cannot
anti-A)
make A or B antigens
o Varying degrees of agglutination by
on their red blood cells
human anti-A,B
- Phenotyped as Group
o Increased variability in the
O
detectability of H antigens, resulting
- Have anti-H (IgM) in plasma:
in strong reactions with anti-H
o Can donate RBCs to any member of
o Presence or absence of anti-A1 in
the ABO group
the serum
o Cannot receive blood from any
member of the ABO group
- Does not react with anti-H lectin
- Genetic Basis:
o Oh is inherited as an autosomal
recessive trait due to a mutation in
the gene FUT1 (H gene) producing a
silenced gene incapable of coding
to the enzyme, a-2-
fucosyltransferase
Weak A Phenotypes o This mutation is also associated with
a silenced FUT2 gene (Se gene) thus
do not elicit ABH expression in
secretions
- Inheritance of ABO but in combination with
hh gene are written as OhA, OhB+, & OhAB
- Oh compatible only with another Oh
Para-Bombay Phenotype
24
- Rare phenotypes in which RBCs are o ‘hangovers’ in Type A blood group

completely devoid of H antigens or have individuals
small quantities of H antigen present o ‘criminality’ in Type B blood group
- RBCs express weak forms of A & B antigens individuals
- Presence of A & B enzyme but no H enzyme o ‘good teeth’ in Type O blood group
(Ah, Bh, ABh) individuals
GENETIC BASIS
Mutates FUT1 (H gene) Silenced FUT1 (H gene)
with or without active with an active FUT2 (Se) • Known to depress antigen strength (seen in FT):
FUT2 (Se gene) gene o Leukemia
- Greatly reduced L- - Production of H, A, o Chromosome 9 translocations
fucosyltransferase B type 1 antigens in o Hemolytic disease
- Low amounts of H, secretions, • Mixed fied or weakened ABO antibody
A, & B antigens including plasma expression (BT):
- Serologically - These antigens may o Hodgkin’s disease
undetectable absorb onto RBC
- Detected using membranes
adsorption & yielding weakly
elution techniques expressed H, A, & B
o Hypogammaglobulinemia
antigens
• Presence of B antigen in a Type A individual:
o Acquired B phenomenon
ABO Discrepancies
• Lack of detectable ABO antigens:
- Occur when unexpected reactions are seen
o Carcinoma of the stomach &
in the forward and reverse grouping
pancreas
- Due to problems in the patient’s red cells
(FT) or the patient’s serum (RT)
Categories of ABO Discrepancies
- May be due to unexpected or missing
GROUP I
reactions
- Discrepancies associated with unexpected
- Must be resolved by Blood Banker prior to
reactions in the reverse grouping
reporting a patient or donor ABO blood
- Presents as weakly reacting or missing
group
antibodies
- More common than the rest of the other
Common causes of technical errors resulting in ABO
discrepancies
discrepancies:
- Cause: Depressed or no antibody production
- Resolution:
o Repeat testing the same sample
Newborns Patient using
o Acquire information regarding
patient’s age, diagnosis, transfusion immunosuppresives
history, medications, & history of Elderly Patients ABO subgroups
pregnancy Hypogammaglobulinemia Bone marrow / Stem
o Draw ne sample cell transplant
- Note: Congenital / Acquired Diluted ABO
o Interpretation of the ABO type must Immunodeficiency States antibodies via
be delayed until the discrepancy is plasma or exchange
resolved transfusion
o Weaker reactions usually indicate
the discrepancy because grouping Resolution in the BB Lab:
reactions are very strong (3+ to 4+)
1. Obtain patient’s data (96 years old patient)

2. Enhance the weak or missing reaction in the
serum
▪ Repeat BT then start with incubating the
patient serum with reagent A & B cells at
room temperature for approx. 15-30
minutes, centrifuge
ABH Antigens & Antibodies in Disease:
• Blood group mythology/misconception:
25
▪ Or by adding 1 or 2 drops more 1+ 4+ 4+ 0

plasma/serum to the test (probably in the Forward Type? AB Reverse Type? B
post zone effect)
▪ If still no reaction, incubate serum-cell Cause?
mixtures at 4°C for 15 minutes → In the B(A) phenotype, the B gene transfers
trace amounts of the immunodominant
GROUP II sugar for the A antigen. Due to elevated
- Associated with unexpected reactions in the expression or overactive nature of B
forward grouping enzyme. These trace amounts are picked up
- Due to weakly reacting or missing antigens or detected by certain clones of
- Least frequently encountered discrepancies monoclonal anti-A antisera
- Examples:
o Subgroups of A / B 1. Determine the patient’s diagnosis & transfusion
o Leukemias history
o Hodgkin’s diseases (some) 2. Test red cells with additional antibody anti-A
o B(A) Phenotype reagents from other manufacturers or a source
o Acquired B phenomenon / pseudo-B of human polyclonal anti-A
antigen
1. Excess amount of blood group-specific solube

(BGSS) substances present in plasma that can
neutralize reagent anti-A or anti-B
(stomach/pancreatic cancer)
2. Antibodies to low incidence antigens found in
Resolution in the BB Lab: reagent anti-A or anti-B antisera
3. Chimerism
▪ Presence of 2 cell populations in an
individual (recognized as self)
▪ Discovered in twins born from a O mother &
B father
▪ Types:
o True chimerism:
▪ found in twins & exist through
lifetime
1. Obtain patient’s data (cancer of the o Dispermy:
colon/rectum) ▪ no history of twin, 2 sperm
2. Test patient’s serum with autologous RBCs (anti- fertilizing 1 egg
B in serum will not agglutinate patient’s RBCs) o Natural chimera:
3. Change pH of anti-B antisera >8.5 or <6.4 (no ▪ in utero exchange of
reaction with anti-B) erythropoietic tissue
4. Treat patient’s RBCs with acetic anhydride to o Temporary chimera:
reacetylate A antigen (this will take out reaction ▪ following blood transfusion of
with anti-B in the FT) compatible but non-blood
specific donor blood (A
patient receives O cells)
GROUP III
1. Obtain patient’s history (patient has leukemia) - Between forward & reverse groupings
2. Incubate test mixture at RT up to 30 minutes - Causing by protein or plasma abnormalities
3. If still negative, incubate the test mixture at 4°C - Result in rouleaux
for 15-30 minutes formation/pseudoagglutination:
o Elevated levels of globulin (MM, WM)
PATIENT’S RED CELL PATIENT SERUM W/ RGT o Advanced cases of Hodgkin’s
WITH RED CELLS lymphoma
Anti-A Anti-B A cells B cells o Elevated levels of fibrinogen
26
o Plasma expanders such as dextran & present on the person’s red

polyvinnylpyrrolidone blood cells
o Wharton’s jelly in cord blood samples
Resolution in the BB Lab:
Resolution in the BB Lab:

✓ Obtain patient history (cold autoimmune / I
blood group)
✓ Forward:
1. Patient RBCs should be incubated at 37°C
for a short period
2. Then wash with saline at 37°C, 3 times, then
retyped
3. If unsuccessful, add 0.01 M dithiothreitol to
disperse cold reacting IgM antibodies
✓ Reverse:
1. Obtain patient info (WM) → Reagent RBCs and patient serum can be
2. Washing patient’s RBCs or cord blood samples warmed to 37°C, mixed, tested, and read at
several times with saline (remove interferences) 37°C
3. Or performing saline dilution or saline
replacement
----------------------------------------------------------------------------
--------
▪ If the forward grouping is affected, wash cells to
remove protein & repeat test
▪ If the reverse grouping is affected, perform
saline replacement technique (more common)
o Cells (reagent) & serum (patient)
centrifuged to allow antigen & antibody
to react (if present)
o Serum is removed & replaced by an
equal volume of saline (saline disperses
cells)
o Tube is mixed, centrifuged, & re-
examined for agglutination (macro &
micro)
----------------------------------------------------------------------------
--------
GROUP IV
Rare Group IV Discrepancies
- Discrepancies between forward & reverse
1. Individual with
groupings due to miscellaneous problems
antibody against
o Cold reactive autoantibodies
acriflavine dye
o Patient has circulating RBCs of more
2. Cis-AB
than 1 ABO group due to RBC transfusion
→ Inheritance of
or marrow/stem cell transplant
both AB genes
o Unexpected ABO isoagglutinins
from 1 parent
▪ Antibodies produced by 1
carried on 1
individual that causes
chromosome
agglutination of cells of other
→ And inheritance
individuals of the same species
of O gene
o Unexpected ABO alloantibodies
inherited from
▪ An antibody formed in response
the other
to pregnancy, transfusion, or
parent
transplantation targeted against
→ Offspring inheriting 3 ABO genes instead of 2
a blood group antigen that is not
→ Express weakly reactive A & B Ag
27
→ Has weak anti-B which reacts to ordinary B Gene products: Glycosyltransferases

antigens but not with cis-AB RBCs Immunodominant H antigen: ______________
sugars: A antigen: ______________
B antigen: ______________
Antigen expression: Cord blood cells: weak
Genetic loci: ABO blood group system:
______________
H system: ______________
Major alleles: A1, A2, B, O
H, h
Bombay phenotype: Genotype: hh; no H or ABO
antigens
Secretor status: Se allele; soluble H & ABO
antigens
Landsteiner’s rule: Serum possess ABO antibody
against the antigen absent
Antibody production: No antibodies detectable in
newborns; decreases with age
CHAPTER SUMMARY Antibody IgM & IgG
Widespread antigen Blood cells, tissues, body fluids, immunoglobulin class:
distribution: secretions In vitro reactions: At or below room temperature
Biochemical Glycolipid / glycoprotein Complement binding: Yes; some hemolytic
composition:
Common structures: Type 1 & Type 2
oligosaccharide chains
28
TESTING THE SECRETOR STATUS USING SALIVA

- Presence of A, B, H substances mediated by the - These individuals are known as secretors and
Se gene on chromosome 19 that codes for the L- their product as substance (Blood Group Specific
fucosyltransferase, which adds a sugar molecule Soluble Substances / BGSS)
(L-fucose) to Type 1 paraglobulin chains found in - Can be detected using antisera but reaction is
secretion: invisible to the naked eye
o saliva, sweat, urine, etc. - These water-soluble substances have the ability
to neutralize or inhibit the capacity of antibodies
to agglutinate RBCs
Principle for the determination of secretor status:
Procedure 1: Collection & Preparation of Saliva 4. Transfer 1mL to another new tube & discard the
1. Collect 2 ml saliva & transfer to tube debris or waste (bottom part of the tube)
2. Place in water bath (100°C) for 10 minutes 5. Make a 1:2 dilution (1 ml of NSS & 1 ml of
o Purpose: to inactivate any enzymes supernatant)
(interferences) **Discard any opaque or semisolid material
3. Take out and centrifuge for 10 minutes
o If still cloudy, reheat for another 5
minutes
Procedure 2: Test for Secretor Status 3. In another set of tubes, label and do the
Reagents: following:
• 5% RBC suspension (A or B)
• 1:4 Known Antisera (A or B)
**AABB: Add in lighter reagents before dark colored Contents:

Diluted Saliva 1 drop 1 drop
1. Prepare a 5% suspension of Known A & B cells Diluted Anti-A 1 drop -
Diluted Anti-B - 1 drop
Mix & cover with Nescofilm, incubate both tubes at RT
for 10 minutes
5% Known A Cells 1 drop -
5% Known B Cells - 1 drop
Mix & cover with Nescofilm, incubate both tubes 30-60
minutes (RT) or 15 minutes @ 37° waterbath
2. Prepare a 1:4 dilution of Known Anti-A & Anti-B
reagents Centrifuge for 15 seconds, gently dislodge, and
examine for agglutination
Grade the results
29
Rh BLOOD GROUP SYSTEM
History:
- Philip Levine & Rufus Stetson (1939):
described a hemolytic transfusion reaction
in an obstetrical patient
1. Based on genetic inheritance:

a. Fisher-Race: DCE terminology
b. Wiener: Rh-Hr terminology
2. Based on the presence or absence of a
given antigen:
a. Rosenfield & Co-workers
(Rosenfield)
3. Based on updated numeric terminology:
a. International Society of Blood
Transfusion (ISBT)
- Landsteiner & Wiener (1940): they happen to
be doing experiments on guinea pigs &
rabbits & decided to inject them with blood **D is the most immunogenic (determines if you’re
coming from the monkey, Rhesus macaque Rh neg or pos)
(Macaca mulatta) **d doesn’t exist but it is used as a ‘filler’ for the
nomenclature
**Antithetical = opposite genes in the red cell
**Allele = opposite genes in the chromosome
A. FISHER-RACE: DCE Terminology

- Ronald Fisher & Robert Race (1940):
postulated that the antigens of the Rh
system were produced by 3 closely linked
set of alleles
- Levine & Co.: - Each gene on chromosome 1 was
o Made another investigation and responsible for producing an antigen &
demonstrated that the same were thus given the same letter designation
agglutinin from his obstetric patient, - Genes that code for antigens:
and the agglutinin described by o D, d (used only as a placeholder, d
Landsteiner & Weiner appear to antigen doesn’t exist)
define the same blood type o C & allele c
o Many years later scientists o E & allele e
recognized that the 2 antibodies - Theory: each person inherits a set of genes
were different & thus renamed to (haplotype) from each parent & express the
avoid confusion corresponding antigens on the RBCs
(codominant)
- Genotype: _ _ _ / _ _ _
Humans can form anti-Rh antibodies & anti-LW

antibodies which confirmed presence of other
antigens other than ABO
Nomenclature
- By the mid-1940s, there were about 5 Rh
antigens recognized and over 57 different
specificities that continue to grow in number
and make up the Rh blood group system
- Major Rh antigens: D, C, c, E, e
- Terminologies for Rh are derived from 4 sets
of investigators:
30
o if h precedes the r = refers to either

small c or e antigens
- Say or example you have a patient whose
probable genotype is DcE/DcE
- Easier to verbally convey to a co-worker
when saying R2R2 instead
C. ROSENFIELD: Alphanumeric Terminology

- Richard Rosenfield & associates (1960):
proposed a system that assigns a number to
each antigen of the Rh system in order of its
discovery to the Rh system or recognized
relationship
- Simply demonstrates the presence or
absence of Rh antigen on the red blood
cells
RARE DEFINITION
- Each antigen is assigned numbers after the
PHENOTYPES
base symbol, “Rh”:
DC-, Dc- If a person lacks E or e antigens
o Rh1: D
D-- If a person lacks all CcEe antigens
o Rh2: C
(D deletion)
o Rh3: E
---/--- If a person has no antigens at all
o Rh4: c
(Rhnull)
o Rh5: e
(D),(C),(e) Weakened expression of Rh - Well suited for electronic processing
antigens (Rhmod)
Rosenfield Nomenclature Rules
B. WIENER: Rh-Hr Terminology - A phenotype on the other hand is expressed
by the base symbol of “Rh” followed by a
colon & a list of the numbers of the specific
antisera for the antigen used
- A minus sign “-“ preceding a number
designates the absence of the antigen
- Example:
- Alexander Wiener believed only 1 gene was o RBCs typed D+, C+, E+, c-, e-
responsible for defining Rh & this gene o Rosenfield translation = Rh: 1, 2, 3, -4,
produced 1 agglutinogen containing a -5
series of 3 blood factors - If an antigen has not been typed, its number
- Eight (8) major alleles: R0, R1, R2, Rz, r, r’, r’’ &ry will not appear in the sequence at all,
- Complex & unwiedly terminology example:
o Nevertheless, many blood bankers o Same typing but RBCs not tested for
use modifications of this e antigen:
nomenclature in combination with o Rosenfield translation = Rh: 1, 2, 3, -4
others as it is easier to verbally
communicate D. ISBT Updated Alphanumeric Terminology
- Formed the Committee on Terminology for
Red Cell Surface Antigens whose main task
was to establish a uniform nomenclature
that is both eye- and machine-readable
- Adopted a 6-digit number for each
authenticated antigen belonging to a
blood group
- Nomenclature:
o First 3 numbers = represent the blood
system
o Remaining 3 numbers = represent
antigen specificity
- The numbers “004” have been assigned to
Rh Blood Group System
ANTIGEN NUMERIC ISBT NUMBER

D RH1 004001
C RH2 004002
E RH3 004003
- Blood factors:
o if r precedes the h = refers to either c RH4 004004
big C or E antigens e RH5 004005
31
Characteristics of Rh Antibodies:
- Most Rh antibodies are IgG
immunoglobulins (37°C)
- IgM antibodies form first but quickly
transition to IgG
- IgA antibodies have been reported but not
routinely tested
- Usually produced following exposure of the
individuals immune system to foreign RBCs
through pregnancy or blood transfusion
- Rh antibodies show dosage (reacting
strongly with RBCs possessing double dose of
Rh antigen):
o Anti-E (3+) against E+e- / Anti-E (1+)
to E+e+
Characteristics of Rh Antigens: - Rh antibodies are enhanced when testing
- Transmembrane proteins (with a with enzyme-treated RBCs
glycosylated base) - According to immunogenicity: D>c>E>C>e
- D deletion phenotype has the most D
antigen sites since there is no competition Clinical Significance of Rh Antibodies:
with other Rh antigens - When Rh antibodies are identified, it is
- Other considerations: important to understand the clinical
o Weak D-positive units are labelled D- implications when Rh incompatibility exists
positive and should be transfused 1. Transfusion Reactions
only to D-positive recipients o Circulating antibodies appear 120
o AABB Standards require testing for days after 1st exposure & within 2-7
weak D on donor red cells that do days after a secondary exposure
not directly agglutinate with anti-D o Rh-mediated hemolytic transfusions
reagents result in extravascular destruction of
antibody-coated RBCs
o DAT is usually positive (+)
o Antibody screen may or may not
detect antibody
o Blood components being transfused
should lack antigen
2. Hemolytic Disease of the Fetus & Newborn
(HDFN)
o Rh causes the most severe form of
erythroblastosis fetalis
Rh Antigen Function:
o Rh antigens are well developed on
- Since they are transmembrane proteins,
fetal cells
they play a role in maintaining the structural
integrity of red cells:
o Westhoff & colleagues: transport
ammonia
o Other hypothesis: CO2 transporter
- Greatest number of D antigen sites: rare Rh
phenotype (D--)
- Greatest number of D antigens (among
commonly encountered): R2R2
Molecular Genetics: RHD & RHCE genes
- Initial theories of Rh genetic control
postulated:
o Fisher-Race Theory =
3 genes (D gene, C/c gene, E/e
gene)
o Wiener Theory = 1 gene (Rh0 gene)
- Today’s most widely accepted theory:
o Dr. Patricia Tippett Theory =
2 genes (RHD & RHCE)
ANTIGEN AMINO ACID POSITION - These genes code for proteins on which Rh
C Serine 103 antigens reside:
c Proline 103 o RHD codes for RhD antigen
E Proline 226 o RHCE codes for:
e Alanine 226 ▪ RhCe antigen
32
▪ RhcE antigen
▪ RhCE antigen
▪ Rhce antigen
Weak D Variations of D antigen expression

- When Rh-positive sample are typed for the
D antigen, one expects a strong reaction,
however, some individuals possess D antigen
Rh Genes that is so weak they require an IAT to detect
- RHD & RHCE genes have 10 exons each & the presence
are 97% identical - History known as Du type based on the
- Rh-positive Phenotypes thought that it was a new antigen, later on
o Codominant inheritance of 1 or 2 proven untrue as no anti-Du is ever
RHD gene/s & 2 RHCE genes produced by the body
o Mutations can occur weak - For many years, individuals were referred to
expression, but individuals still generally as weak D
considered Rh+
- Rh-negative Phenotypes
o Arise from 3 diffferent mutations
falling into 3 ethnic backgrounds:
▪ European (deletion of RHD
but possess 2 RHCE gene)
▪ African ( RHD pseudogene;
identical to RHD but has
mutation so still no RHD
protein)
▪ Asian (mutation in RHD gene
causing a person to be typed
as Del when, Rh-neg)
Mechanisms of weak D:
a. C trans to RHD
RhAG Gene
- Described as a position effect or gene
- Rh antigens reside on transmembrane
interaction effect (steric arrangement)
proteins integral to RBC membrane coding
which appears to interfere with D antigen
for 416 amio acids that traverse 12 times
expression
- Rh-associated glycoprotein (RhAG;
- Dce/dCe
Coexpressor):
- D antigen is structurally complete
o Coded for by the RHAG gene
- But patient may type as Rh-negative, so
o Resides on chromosome 6
confirm with IAT
o Similar in structure to Rh proteins but
- Individuals can receive D-positive RBCs with
glycosylated
no adverse effects
o Must be present for Rh antigen
expression
b. Weak D
o If by itself does not express any Rh
- D antigens appear to be complete but
antigens
fewer in number due to mutations in the RHD
gene
- Changes happen inside the red cell
membrane
- Patient may type as Rh-negative or weakly
reactive
- No anti-D is produced since as far as the
immune system is concerned, these
changes cannot be seen
Patient can be safely given Rh-positive blood (+)

33
c. Partial D (D Mosaic) A. Rhnull

- Occurs when 1 or more D epitopes within - Indivuals who lack ALL Rh antigens on their
the entire D protein is either missing or RBCs
genetically altered - 2 types;
- Results to a weaker than expected 1. Regulator Type
serological action or no reaction at all ➢ Mutation occurs in the RHAG
- Individuals are D-positive but produce anti- gene
D that react with other D-positive cells, ➢ No RhAG protein expression
except their own ➢ Thus, no RhD or RhCE expression
- Anti-D causes HDFN & HTR ➢ Even when individuals have
- Protein changes occur external to the red normal RHD & RHCE genes
cell membrane (RHD-RHCE-RHD) ➢ Individuals can still pass on
- Individuals make antibody to the portion of normal RHD & RHCE gene
the RHD gene that they are missing **patients suffer from mild compensatied hemolytic
- Rh-negative blood should be used for anemia & stomatocytosis. Transfuse only with Rhmod
transfusion blood
2. Amorphic Type
➢ RHAG gene is normal but….
o Mutation in each of the
RHCE genes
o And deletion of the RHD
gene
**Individuals regardless of type are negative for

high prevalence antigen LW & for FYS. May also
have depressed S, s, & U antigens
B. Rhmod
- Has a partial suppression of RH gene
- Caused by: mutations in the RHAG gene
d. Del - Results to weakened expression of normal
- Phenotype occurring in individuals whose Rh & LW antigens
red blood cells possess an extremely low - Example: (D), (C), (e)
number of D antigen sites that most reagent - Can exhibit other blood group antigens on
anti-D are unable to detect the red blood cell
- Way to detect: adsorption & elution - But may also have depressed S, s, & U
technique antigens
o Incubate anti-D reagent with patient - Clinical symptoms are usually less severe &
RBC (D-antigen positive +) rarely clinically remarkable
o Maintain incubation at 37°C for 1
hour, centrifuge, remove serum RH Typing Reagents
o Wash RBCs at least 5 times then add - May be derived from a variety of sources:
equal amount of saline 1. Saline anti-D
o Then elute (56°C, 10 minutes) the 2. High protein anti-D
anti-D off the adsorbed red cells 3. Monoclonal anti-D
4. Monoclonal anti-D
5. Chemically modified anti-D
- Requirements:
o Reagents consists primarily of IgG
anti-D
o Nonhuman derived to decrease risk
of transmitting infectious disease
e. D epitopes on RHCE o Monoclonal & Polyspecific so
- Results from specific amino acid in the RHCE reagent can recognize multiple
gene resulting in an RhD epitope on the epitopes of the RHD protein
RhCE protein
- Show positive reactivity even though D
epitope is on the RhCE protein
- Examples: DHAR (R0HAR) & ceCF (Crawford)
phenotype
- Classified as RhD-negative since individuals
essentially lack D protein
- Should only be RhD-negative blood
Rh Deficiency Syndrome: Rhnull & Rhmod

34
Rh TYPING
Blood Collection: 5. D epitopes on RHCE: inserts RhD epitopes to RhCE
- Each person should pick a partner to collect protein
blood from & determine the Rh type (EDTA tube) **Individuals with weak D (D u) variants are considered
- After collection, prepare a 4% RBC suspension of ‘Rh-positive’ as donors and ‘Rh-negative’ as recipients in
patient cells using 3 ml total volume routine testing.
**To avoid HTR if patient might have Anti-D/weak D
variant confirmed through molecular testing/family
studies
Procedure:
1. Prepare and label the following:
Anti-D Negative Control
Anti-D 1 drop -
2% BSA/LISS - 1 drop
4% Patient RBCs 1 drop 1 drop
Rh-positive: D antigen (most immunogenic)
Rh-negative: no D antigen 2. Mix gently and cover with Nescofilm
Chromosome: 1p (short arm) 3. Centrifuge for 15 seconds
ISBT Number: 004 (4th blood group discovered) 4. Gently dislodge red cell button and examine for
agglutination under bright light
Genes that code for Rh antigens: 5. Grade and record each reaction. Submit tubes
1. RHD: D antigen together with output for checking.
2. RHCE: C, c, E, e antigens
**If appears to be Rh-negative, proceed to confirmatory
Nomenclatures: test (Weak D Test) before releasing results
1. Fisher-Race: DCE Terminology
2. Wiener: Rh-Hr Terminology Confirmation of Rh-negative Result:
3. Rosenfield: Alphanumeric Terminology AHG Test for Weak (Du Test)
4. ISBT: Updated Alphanumeric Terminology - Rh negative results should not be readily reported
unless confirmed by Weak D test
Immunogenicity: D > c > E > C > e - Weak D expression can recognized most reliably
by an IAT after incubation of the test red cells
Rh-Associated Glycoprotein (RhAG) with Anti-D
- A.k.a. “Coexpressor”
- Chromosome 6 Procedure:
1. Using same 4 ml patient 4% RBCs initially typed as
Rh Antibodies Rh-negative label and prepare the following:
Anti-D Negative Control
- IgG, 37°C, show dosage
Anti-D 1 drop -
- Causes HDFN (most severe)
2% BSA/LISS - 1 drop
4% Patient RBCs 1 drop 1 drop
Other Rh Phenotypes:
2. Mix gently, cover with Nescofilm. Incubate for 15
1. D-deletion: D - -
minutes, water bath (37 °C)
2. Rhnull: - - - / - - -
3. Wash cells 3x with NSS. Decant saline completely
3. Rhmod: Example(D)(C)(e)
after washing
4. Add in AHG: 2 drops on each tubes
Typing reagent:
5. Mix gently, cover with Nescofilm, centrifuge for 15
- Anti-D
seconds. Dislodge.
- Nonhuman, monoclonal, polyspecific
6. Examine for agglutination and interpret results:
o (+) agglutination: Weak D Variant
Weak D Variants: (refer to lecture notes)
o (-) agglutination: Rh negative?
1. C trans to D: steric arrangement/hindrance
7. If no agglutination, confirm true Rh-negative cells
(opposite side)
using Coombs’ check cells, place 1 drop to each
2. Weak D: doesn’t produce Anti-D
tube and examine for agglutination:
3. Del: Phenotype occurring in individuals whose red
o (+) agglutination after check cells:
blood cells possess an extremely low number of D
True Rh-negative
antigen sites that most reagent anti-D are unable
o (-) agglutination after check cells:
to detect (elute to detect)
Test improperly done, repeat test
4. Partial D / D-mosaic: 1 or more D epitopes within
the entire D protein (either missing or genetically
altered)
35
MINOR BLOOD GROUPS

Reason for studying other blood group systems: - Antibodies: described by their antigen
- ISBT has defined 36 blood systems notation with he prefix anti-, before the
- More than 300 unique antigens have been antigen symbol (don’t forget the hypen)
documented on RBCs - Numeric terminology: introduced by ISBT
- Knowledge of other blood group systems assigned 3 numbers to each blood group
provides the foundation for solving complex system
antibody problems in a logical and efficient - Blood Group Collections: antigens that have
manner a biochemical, serologic, or genetic
- ISBT: ”A blood group system (BGS) consists of relationship but do not meet the criteria for
one or more antigens controlled at a single a system (assigned # 200)
gene locus, or by two or more very closely - All remaining RBC antigens not associated
linked homologous genes with little or no with a system or collection are catalogued
observable recombination between them.” into the 700 series (low prevalence antigens;
- The antigens are sorted into blood group <1% of the population) or 901 series (high-
systems, blood collections, and series of prevalence antigens; >90% of the
independent antigens population)
Functional roles of some BGS: LEWIS BLOOD GROUP (007)

BIOLOGICAL ROLE BLOOD GROUP SYSTEM
Glycosyltransferases ABO, H, Lewis, P1Pk
Structural MNS, Diego, Gerbich
relationship to the
red blood cell
Transport proteins Rh, Kidd, Diego, Kx
Complement Chido/Rodgers, Cromer, Knops
pathway molecules
Adhesion molecules Lutheran, Xg, Landsteiner-
Wiener, Indian
Microbial receptors P, Lewis, MNS, Duffy, Cromer
Biologic receptors Duffy, Knops, Indians
Terminology:
- Genes: written in italics or underlined when
italics are not available (E.g. when
handwritten)
- Allele number/letter (always superscript)
- Some antigens have numbers, superscript letters **Enzymes (proteolytic) are used as
- Antigen names: written in regular type confirmatory; Not all blood groups can
without italics underlining be cleaved by enzymes
- Phenotype: a description of which antigens
are present on an individual’s RBCs and - Mourant (1946): reported from a
simply indicates the results of serologic tests first patient to form the antibody of this
on those RBCs, their numbers written blood group
sometimes with a subscript - Not intrinsic to RBC but are on type 1
o E.g.: A1 (antigen), A1 (phenotype), glycosphingolipids from the plasma that are
A1 (allele) passively adsorbed onto the RBC
- For Letter antigens: a plus/minus sign written membrane
on the same line as the antigen used to o Can be found or formed in the
designate the presence or absence of the plasma & will adhere to RBC
antigen - Manufactured by tissues cells & secreted
o E.g.: N+ & S- into body fluids (not an integral part of RBC)
- For antigens with superscripts written as - Development begins first week after birth &
phenotypes: the letter of the superscript is may continue for 6 years
placed in parentheses on the same line as - H & Se genes code for L-fucosyltransferase
the letter defining the antigen - Transfers L-fucose to terminal galactose of
o E.g.: Fya(a+) & Jk (b-) precursor structures
- For antigens with a numerical designation: - H gene acts on Type 2 precursors, creating H
the letter defining system is followed by a antigens on RBCS
colon then the number representing the - Se gene acts on Type 1 precursors, creating
antigen H substance in secretions
o E.g.: Rh: -1, 2
- Genotype: composed of the actual genes
that an individual has inherited and can be
determined only by family or DNA studies
(molecular testing)
36
Biochemistry of Lewis Antigens:

- Lewis determinants are carbohydrates on
o Glycolipids: cell-bound antigens
Inheritance of Lewis Antigens: absorbed from plasma
- Dependent on 3 genes to produce Lewis o Glycoproteins: saliva, not adsorbed
antigen structures: on RBC
o H (FUT1) - NOT EXPRESSED ON CORD CELLS
o Se (FUT2) - Lewis antigens can be lost from the red cell
o Le (FUT3) (dislodged) in:
- Gene products are glycosyltransferases o IM
- Specifically, L-fucosyltransferase o Alcoholic cirrhosis
o Adds L-fucose to precursor structures o Pancreatitis
at specific locations o Pregnancy (↑ plasma volume)
- The h, se, le genes are amorphs and - Leb antigen: receptor for Helicobacter pylori
produce no detectable product - GIT: primary source of Lewis glycolipids
- If the Le gene is inherited, Lea antigens are
formed
- If both the Le gene & Se gene are inherited,
Leb antigens are formed instead
LEWIS PHENOTYPES Development of Lewis Antigens:

• Le (a+b-) RBCs are from ABH nonsecretors - Lea & Leb glycoproteins can be present in
• Le (a-b+) RBCs are from ABH secretors the saliva of newborns
• Le (a-b-) RBC phenotype are either: - Lewis glycolipids are not detectable in
o Secretors (80%); or plasma until about 7-10 days after birth:
o Nonsecretors (20%) o Infant cord blood phenotype as Le
• Le (a+b+) RBC phenotype is rare among (a-b-) at birth & later transform to Le
whites & Africans but is frequent in Asians (a+b-) or Le (a-b+)
(weak Se gene) - lele genes phenotype Le (a-b-) at birth and
• Lea antigen cannot be converted into Leb for the rest of their lives
antigen because of steric hindrance from
the fructose added to make Lea Lewis Antibody Characteristics:
• Leb individuals although they phenotype as - are IgM (RT/Colder) and have no clinical
Le (a-b+) will always have some significance
undetectable Lea antigens - antibodies occur almost exclusively in the
serum of Le (a-b-) individuals, usually
without red cell stimulus
- Le (a-b+) individuals do not produce anti-
Lea
-
37
- Le (a+b-) individuals do not produce anti-

Leb (rare)
- Le (a-b-) individuals can produce both anti-
Lea & anti-Leb
GENES ANTIGENS IN PHENOTYPE

PRESENT SECRETIONS Ii INHERITANCE
Le sese H Lea Le (a+b-)
Le Se H Leb H Le (a-b+)
Le (undetected)
a
lele sese H None Le (a-b-)

lele Se H H Le (a-b-)
Le sese hh Lea Le (a+b-) - The I and i antigens exist on the precursor
Le Se hh Lea Le (a+b-) paraglobuside chains at a position closer to
lele sese hh None Le (a-b-) the red cell membrane
lele Se hh None Le (a-b-) - i antigen
o Produced from a sequential action
Anti-Lea Anti-Leb of -3-N-galactosyltransferase
▪ Most commonly ▪ Not as common or o Linear chains
encountered generally as strong - I antigen
▪ IgM, detected at RT as anti-Lea o Caused by branching
▪ Not clinically ▪ Usually IgM, can -6-N-acetylglucosaminyltransferase
significant bind complement that attaches a GlcNAc1 → 6
▪ But if sometimes ▪ Divided into: linkage from Gal
reactive at 37°C → Anti-LebH = o Branched chains
▪ Should not be react with Leb +
ignored if reactive H Biochemistry of Antigens:
as it may cause → Anti-LebL = react - i antigen converts into the I antigen over a
rare HTRs with any Leb 2-year period
▪ Give Lea-negative antigen - Both antigens are present on glycolipid &
blood to patient regardless of glycoprotein structures on the red cell
ABO type membrane
- Also found on membranes of leukocytes &
- Challenge to identify because agglutination platelets
is observed at IS, 37°C, AHG but - Can also be found as soluble glycoprotein
agglutination is fragile and easily dispersed antigens in plasma & in body secretions
- Reactivity of Lewis antibodies can be such as:
greatly enhanced with enzyme-treated o Human milk
RBCs o Amniotic fluid
- Lewis antigens are resistant to treatment with o Ovarian cyst fluid
DTT & glycine-acid EDTA
- Hemolysis may be seen in fresh serum due
to presence of Ca++ which activates
complement & binds to anti-Lea (IgM)
I BLOOD GROUP (027)
- Composed of only 1 antigen, I, which was

assigned to a blood group system in 2002
- Product of I gene (IGnT/gcnt2) is: N-
acetylglucosaminyltrsnsferase
- I antigen is expressed on adult cells
- The i antigen remains in the Ii blood group
collection 207
- Gene for i antigen not been found
- The i antigen is expressed on newborn & Ii ANTIGENS & ANTIBODIES
cord blood cells - Both I & i are high prevalence antigens but
- I (027001) & i (207002) are not antithetical are expressed in a reciprocal relationship
antigens - At birth:
o Infant RBCs are rich in i antigen
o I is undetectable
38
- First 18 months of life: quantity of i slowly

decreases and I increases Antibodies to compound antigens:
- Adult RBCs:
o Rich in I antigens
o Have only trace amounts of i
antigens
- No true I- or i- phenotype.
o But some people never change their
i status after birth, these people are
called “adult I” (recessive) and can - Many other I-related antibodies have been
therefore appear as if he or she is I-. described:
o Anti-IA, -IB, -IAB, -IH, -iH
- These antibodies react to compound
antigens (combination of 2 antigens that
must be present on the RBC for the antibody
to be reactive)
- The I & I antigens are resistant to treatment
with DTT & glycine-acid EDTA
THE IT ANTIGEN & ANTIBODY

I Antibody Characteristics:
- Anti-I is found as a cold-reacting, clinically
insignificant IgM which may require testing
at 4°C or against enzyme-treated RBCs to
be detected
- Found in the serum of many normal, heathy
individuals, but not associated with in-vivo
RBC destruction - Curtain et al. (1965): reported a cold
- Anti-I binds complement, polyspecific AHG agglutinin in Melanesians that did not
can detect C3d component that may demonstrate classical I or i specificity
attach or be left behind on the RBC - Booth & coworkers described the agglutinin
- Problem with Anti-I: autoantibody frequently a year later:
encountered in the lab causing o Antibody had strong reactions with
discrepancies (cold autoantibodies) cord RBC
- Best way to avoid a suspected cold o Antibody had weak with normal
agglutinin is to never let the antibody react adult RBC
(avoid in vitro RT agglutination) o Antibody most weakly with adult i
- Remedy: prewarming technique (warm RBC
saline 37°C) - Concluded that the agglutinin recognizes a
transition state of i into I and designated it:
Confirming presence of Anti-I: o IT (T for “transition”)
- One way to confirm presence of sample o Anti-IT also IgM
suspected to have anti-I is through the use
of enzyme reagents:
ENHANCED DECREASED UNAFFECTED

ABO-related MINS System Kell System
o ABO/H Duffy System
systems
o Lewis systems
o I system
o P1PK/GLOB
Rh System
Kidd System
39
P, I & MNS Blood Group System ▪ Luke Antigen is also formed

*BGS that Produce Cold Antibodies = PILMNS from this 2nd Pathway but the
- P BGS mechanism is yet to be known
- I BGS
***4-Alpha-Galactosyltransferase
- Lewis BGS
- Gb3 Synthase or Pk Synthase
- MNS (referred to as 1 BGS but only Anti-M is a
- Forms Pk Antigen from Precursor Substance
Cold Antibody)
***3-Beta-N-Acetylgalactosaminyltrasferase
P Blood Group System
- Gb4 Synthase
- Not a Single Blood Group System but 3
- Coded by B3GALNT1 Gene
Systems Interrelated to One Another:
- Converts Pk to P Antigen
o P1 Pk = ISBT 003
o P [GLOB] = ISBT 028 P BGS Phenotypes: (Memorize)
o Luke [LKE] and PX2 [GLOB Collection] Possible Antibodies
Phenotypes Antigens
= ISBT 209 Produced
P1 and P
- Introduced by Landsteiner and Levine in 1927 P1 None
Antigens
- Genes: P2 P Antigen Only Anti-P1
o P1 Pk = Chromosome 22 p No P, P1 nor Pk Anti-PP1Pk
o P = Chromosome 3 P1 and Pk
P1k Anti-P
- Antigens: Antigens
P2k Pk Only Anti-P1 and Anti-P
o P1
***Note that Pk is no longer mentioned in P1 and P2
o P
since it is the precursor of P Antigens so it is
o Pk = AKA Globotriaosylceramide
understood to be present if P is present.
(Gb3) = Converted to P Antigens
***P2 Phenotype = Capable of Producing Anti-P1
o Luke (LKE)
since it does not have P1 Antigens
- Found in RBCs, Plasma and Other Tissues but
***P Null (p Phenotype) = Slightly More Common in
Not Found in Secretions
Japan, North Sweden and in an Amish Group in Ohio
- P1, P and Pk = Found on RBCs, Lymphocytes,
P1 Antigens
Granulocytes and Monocytes
- Poorly expressed at birth and may take up to
- P = Found on Platelets, Epithelial Cells and
7 years to be fully expressed
Fibroblasts
- Antigen strength varies from one individual to
- Resistant to Ficin, Papain, Dithiothreitol (DTT),
another
Chloroquine and Glycine-Acid EDTA
o Some may be P1+s (Strong) or P1+w
Biochemistry of P Antigens: (Weak)
- RBCs and Plasma = Exist as Glycosphinolipids o Blacks have stronger expression of P1
- Hydatid Cyst Fluid = Exist as Glycoproteins than Whites
- Common Precursor Substance of All P - Inhibitor Lutheran Gene = In(lu) = Inhibits
Antigens = Lactosylceramide (Gb2 or Expression of P1 Antigens = Inheritance of this
Ceramide Dihexose) gene leads to P1- even if person is originally
o Gives rise to formation of P1 and P1+
Paragloboside - Deteriorates Rapidly on Storage
o Paragloboside = Type 2 Precursor for o Older RBCs = May Produce False
ABH Antigens and P1 Antigens Negative Reactions to Anti-P1
o Also gives rise to Pk, P and Luke - Also found in Hydatid Cyst Fluid caused by
Antigens Echinococcus granulosus and Patients with
- 2 Distinct Biosynthetic Pathways of P Fascioliasis (Bovine Liver Fluke Disease)
Antigens:
Anti-P1
o Lactosylceramide (Gb2) forms
- Naturally Occurring IgM found most often in
Lactotriaosylceramide which forms
P1- Individuals (P2 Phenotype)
Paragloboside which can give rise to
- Weak, Cold-Reactive Saline Agglutinin that
both P1 and ABH Antigens
Optimally Reacts at 4C
o Lactosylceramide (Gb2) + 4-Alpha-
- Not Clinically Significant
Galactosyltransferase = Pk Antigen
- Never causes HDFN but may cause
▪ Pk Antigen + 3-Beta-N-
Occasional HTR
Acetylgalactosaminyltrasfera
o Cannot cause HDFN since:
se = P Antigen Globoside
▪ IgM Type = Cannot Cross
Placenta
40
▪ P1 Antigen = Poorly Expressed - Described by Tippett from Serum of Patient w/

at Birth Hodgkin’s Lymphoma
- If Anti-P1 is suspected, detect by Incubation - Anti-LKE reacts w/ All RBCs except 2% of P1
at Room Temp or Lower or by Pretreatment of and P2 Phenotypes and those w/ p and Pk
RBCs with Enzymes Phenotypes
- Neutralized or Inhibited by Hydatid Cyst Fluid - All p and Pk Individuals are Luke-
(which contains Soluble P1 Antigens) in the
Disease Associations:
lab to reveal other significant antibodies
Anti-P1 = Parasitic Infxns (Echinococcus
Anti-PP1Pk granulosus and Fascioliasis)
- Formerly known as Anti-Tja Anti-P and Anti-PP1Pk = Spontaneous and
- First described in serum of Mrs. Jay suffering Early Abortions
from a Tumor (Adenocarcinoma) Autoanti-P = Cold Paroxysmal
- Produced by p individuals and is Naturally Hemoglobinuria
Occurring P1, P, Pk and LKE = Receptors for P-Fimbriated
- Each component (Anti-P, Anti-P1 and Anti- Uropathogenic E. coli
Pk) may be separable through Adsorption o Pk = Receptor for Shiga Toxins of S.
- May be of IgM or IgG Forms dysenteriae and Enterohemorrhagic
- Reacts over a Wide Thermal Range and Binds Strain of E. coli = Also a Receptor for
to Complement making them Potent Streptococcus suis
Hemolysins o P = Receptor for Parvovirus B19
- Causes Severe HTR and HDFN
End of P BGS
- Associated with Increased Incidence of
Spontaneous Abortions I Blood Group System
- Composed of 2 High Prevalence Antigens:
Anti-P o I = 027
- Alloanti-P o i = 207
o Naturally Occurring Found in Sera of - Discovered by Wiener and named as I for
Pk Individuals (P2k Phenotype) “Individuality”
o Potent Hemolysin similar to Anti-PP1Pk - Antibody discovered by Wiener reacted with
that reacts to all RBCs except those most blood specimens
with p phenotype and Pk phenotype - I and i are NOT ANTITHETICAL ANTIGENS
o Associated with Habitual Early o I = Branched Structure = Found in
Abortion Adults
- Autoanti-P o i = Linear Structure = Found in
o Causative Agent of Cold Paroxysmal Newborns and Cord Cells
Hemoglobinuria o i is converted to I as a person grows
▪ First seen in Patients w/ - I = Encoded by IGnT Gene = Chromosome 6
Tertiary Syphilis - i = Unknown but not Controlled by
▪ Now more commonly seen Chromosome 6
Secondary to Children with - Formed by the action of Glycosyltransferases
Viral Infection and Formed on the Same Type 2 Precursor
o IgG Type and is a Biphasic Hemolysin Substance (Paragloboside) of ABH in RBCs
▪ At Cold = Antibody Binds to - Ii = Also Found on WBC and Platelet
RBCs Membrane, Other Tissue Cells, Plasma and
▪ At 37C = Causes Complement Secretions (Saliva, Human Milk, Aminotic
Activation and Subsequent Fluid, Urine and Ovarian Cyst Fluid)
Hemolysis
o AKA Donath-Landsteiner Antibody Anti-I Anti-i Anti-IT
o Does not react to routine tests and is Adult I Strong Weak Weak
Demonstrable only with the Donath- Cord i Weak Strong Strong
Landsteiner Test Adult i Weak Strong Weakest
***ABH, P and I Antigens are closely related

- Birth = Infant RBCs are rich in i while I is
biochemically so formation of Compound Cold
undetectable
Antibodies such as Anti-IP1, Anti-iP1 and etc. are
- First 18 Months = Quantity of i slowly
possible.
decreases while I increases
Luke (LKE) Antigens
- Adult RBCs = Rich in I and only Traces of i left
41
- Both I and i varies from individual to individual o IgM or IgG Type and found in serum
- Some people do not convert Newborn i to of Adult i Phenotypes
Adult I and thus becomes Adult i. o Not Clinically Significant since it does
- I and i Antigens not react with Autologous RBCs and is
o Enhanced by Ficin and Papain Not Reactive at 37C
o Resistant to Dithiothreitol and ▪ Compatible Adult i blood is
Glycine-Acid EDTA still donated to these
individuals even if such is
Biochemistry of Ii Antigens:
unnecessary
- I and i Antigens = Precursors for Synthesis of
o Strong Autoanti-I may mimic Alloanti-
ABO and Lewis Antigens = Internal Structures
I and if enough Antibodies bind an
of these Antigens
Individual’s RBC = May be falsely
- Ii = Carried on Type 2 Precursor Substance
typed as I Negative
- i = At least 2 Repeating N-Acetyllactosamine
[Gal(Beta1-4)GlcNAc(Beta1-3)] in Linear Anti-i
Form - Alloanti-i has never been described
- I = Branched Form of i Antigen - Autoanti-i = Fairly Rare Antibody that Reacts
o IGnT Gene (GCNT2) = Encode for N- Strongly with Cord RBCs and Adult i RBCs and
Acetylglucosaminyltransferase = weak with Adult I RBCs
Adds N-Acetylglucosamine to form - Of IgM Type and React Best with Saline-
Branches Suspended Cells @ 4C
- Only seen associated with Infectious
Mononucleosis (Epstein-Barr Virus) and
Lymphoproliferative Disorders
- Seldom causes HTR and IgG Anti-i may cause
HDFN
IT Antigen and Antibody

- Curtain discovered a Cold Agglutinin in
Melanesians that did not demonstrate
Classical I or i Specificity
- Agglutinin (IgG or IgM) reacted Strongly w/
Cord RBCs, Weak w/ Adult I RBCs and Most
Anti-I
Weakly w/ Adult i RBCs
- Benign Cold Autoanti-I, Pathologic Cold
- Agglutinin probably recognizes a transition
Autoanti-I and Alloanti-I
state of i to I and designated as IT for
- Benign Cold Autoanti-I
“Transition”
o Common autoantibody found in
- Occurs frequently in Melanesians and
virtually all sera
Yanomama Indians in Venezuela
o Requires testing at 4C or w/ Enzyme
- Has Special Association w/ Hodgkin’s
Treated RBCs to detect
Disease
o Of IgM Type
o Clinically Insignificant Disease Associations of Ii Antigens and Antibodies:
o May cause Problems in Pretransfusion - Anti-I = Cold Agglutinin Disease and
Testing Mycoplasma pneumonia
o How to Prevent: - Anti-i = Infectious Mononucleosis
▪ Avoid Room Temperature - Anti-IT = Hodgkin’s Lymphoma
Testing - Increased i Antigen on RBCs =
▪ Use Anti-IgG instead of Dyserythropoiesis (Especially CDA Type II or
Polyspecific AHG HEMPAS)
▪ Cold Autoabsorption
***HEMPAS = Hereditary Erythroblastic Multinuclearity
- Pathologic Cold Autoanti-I
w/ Positive Acidified Serum Test
o Causes Cold Agglutinin Disease
- Adult i Phenotype in Asians = Associated with
o Reacts w/ Adult and Cord RBCs @
Congenital Cataracts
Room Temperature and 4C
o Stimulated by Microorganisms with I- End of I BGS
Like Antigens on Surface such as
Mycoplasma pneumoniae
- Alloanti-I
42
MNSsU Blood Group System o However, it can be destroyed by

- Discovered by Landsteiner and Levine ZZAP (DTT + Ficin/Papain)
- ISBT 002 - Neuraminidase = Cleaves Sialic Acid =
- U Antigen = Discovered by Weiner = U for Reduces M and N Reactivity
Universal = High Prevalence Antigen = Found
S and s Antigens:
in 100% of Whites and 99% of Blacks
- Found on Glycophorin B
- Common Haplotypes in Whites in Decreasing
- Antithetical Antigens
Frequency:
- Differ from their Amino Acid Residues at
o Ns > Ms > MS > NS
Position 29:
- Encoded on Chromosome 4
o S = Methionine
o GYPA = Glycophorin A = M and N
o s = Threonine
Antigens = 7 Exons
- GPB is less in amount than GPA
o GYPB = Glycophorin B = U, S and s
- S and s Antigens are located Farther Down
Antigens = 5 Exons + 1 Noncoding or
GPB
Pseudoexon
o Less Easily Degraded by Enzymes
o GYPE = Does Not Make a
- Ficin, Papain, Bromelin, Pronase and
Glycoprotein on RBC Surface but
Chymotrypsin destroys S and s Antigens
Participates in Gene Rearrangements
- DTT, Chloroquine, Glycine-Acid EDTA and
that Result to Various Alleles
Trypsin do not destroy S and s Antigens
o Inherited as Codominant Alleles
- Glycophorin
o Integral/Transmembrane Protein that ***All MNSs Antigens show Dosage Effect meaning:
traverses Whole RBC Surface - Stronger Reactions if Homozygous such as
o NH2 (Amino) Terminal End = Lies (M+N-) or (M-N+)
Outside RBC Membrane - Weaker Reactions if Heterozygous such as
o COOH (Carboxyl) Terminal End = Lies (M+N+)
Inside RBC Membrane
o Glycophorin A = Associates with Anti-M and Anti-N:
Protein Band 3 = Affects Expression of - Naturally Occurring Saline Agglutinins that
Wrb Antigen of Diego Blood Group react below 37C
o Glycophorin B = Associated with Rh - 50-80% IgG while the rest are IgM
Protein and Rh-Associated - Does not bind to complement
Glycoprotein = Greatly Reduced S - Not Clinically Significant and Cannot cause
and s Antigens in Rh Null Individuals HTR and HDFN
o GPA and GPB = Expressed on Renal - More Common in Children than Adults
Endothelium and Epithelium - Particularly More Common in Patients w/
o Absence of GPA and GPB = Not Bacterial Infections
Associated w/ Disease or Decreased - Anti-M = Reacts best at pH 6.5
RBC Survival - Anti-N = Less Common than Anti-M = Seen in
Renal Patients Dialyzed on Equipment
M and N Antigens: Sterilized with Formaldehyde regardless of
- Found on Glycophorin A (Major RBC Sialic their MN Type
Acid-Rich Glycoprotein) o Formaldehyde alters M and N
- Antithetical Antigens Antigens so that the body recognizes
- Differ from their Amino Acid Residues at them as foreign.
Positions 1 and 5:
o M = Serine at Position 1 and Glycine Anti-S and Anti-s:
at Position 5 - IgG that is Reactive at 37C and w/
o N = Leucine at Position 1 and Antiglobulin Test
Glutamic Acid at Position 5 - Binds to complement
- Well Developed at Birth - Clinically Significant and causes Severe HTRs
- M and N Antigens are located at the Outer w/ Hemoglobinuria and HDFN
End of GPA - If Optimal Reactivity of Some Anti-S and Anti-
o Easily Destroyed by Ficin, Papain, s is between 10-22C:
Bromelin, Trypsin and Pronase o Perform Saline Indirect Antiglobulin
- Resistant to Dithiothreitol (DTT) Alone, Test (IAT)
Chloroquine and Glycine-Acid EDTA o Incubate tests at RT and Perform IAT
w/o Incubation at 37C
43
- Seen less often than Anti-M Anti-EnaTS = Reacts w/

▪
Trypsin-Sensitive Area on GPA
U Antigen (Universal):
▪ Anti-EnaFS = Reacts w/ Ficin-
- Always present when S and s Antigens are
Sensitive Area on GPA
inherited
▪ Anti-EnaFR = Reacts w/ Ficin-
- High Prevalence Antigen found in Almost All
Resistant Area on GPA
Individuals
o Anti-Ena = Capable of Causing
- 85% of (S-s-) Individuals are also U Negative
Severe HTR and HDFN
- U-Negative Cells are only found in 1% of the
3. Mk Phenotype
Black Population and is NEVER FOUND IN
o Silent Gene or Null Phenotype of MNS
WHITES
o Deletion of Both Glycophorin A and B
- U-Negative Individuals may only receive U-
o Results to Total Absence of MNSsU
Negative Blood
Antigens
- Resistant to Enzyme Treatment
o Typed as M-N-S-s-U-En(a-)Wr(a-b-)
Anti-U: o Associated w/ Decreased RBC Sialic
- IgG that Reacts w/ either S+ or s+ RBCs Acid but Increased Glycosylation of
- Usually produced by S-s- Individuals since 85% Bands 3 and 4.1
of these individuals are also U Negative
Disease Associations:
- Causes Severe HTR and HDFN
1. 𝑮𝑷𝑨𝑴 = Receptor for Pyelonephritogenic
Strains of E. coli
2. GPA and GPB = Alternative Receptors of
Plasmodium falciparum
MNSsU Antibodies:
Clinically Insignificant = Anti-M and Anti-N
Clinically Significant = Anti-S, Anti-s, Anti-U and Anti-
Ena
Rare MNS Phenotypes:

1. U- Phenotype
o Partial or Complete Deletion of GYPB
o Typed as S-s-U-
o Deletion of U also leads to Deletion of
S and s
o Produced Anti-U in Response to
Transfusion and Pregnancy
2. En (a-) Phenotype
o Ena = Envelope = High Prevalence
Antigen
o All En (a-) Individuals are also M-N- w/
Reduced Sialic Acid on RBCs
o Deletion of GYPA causing Absence of
Glycophorin A and also M and N
Antigens
o 2 Types of En (a-):
▪ En (a-) Fin = Based on a
Finnish Report = Homozygous
Deletion of GYPA
▪ En (a-) UK = Based on an
English Report = Heterozygous
Deletion
o Produces Anti-Ena = Reacts to All
RBCs except En (a-) Individuals
44
Kell, Kidd, Duffy and Lutheran Blood Group System o Kell Antigen Expression depends
*Systems that Produce Warm Antibodies = Kell, Kidd, upon presence of the Kx Antigen
Duffy, Lutheran o Absence of Kx Antigen = McLeod
Phenotype
Kell Blood Group System (ISBT 006)
Biochemistry of Kell Antigens and Kx Antigens:
- Consists of 32 High Prevalence and Low
Kell Glycoproteins
Prevalence Antigens
- RBC Membrane Glycoproteins where Kell
- 1st Blood Group to be Discovered after
Antigens are attached
Introduction of AHG Testing
- Member of the Neprilysin (M13) Family of
- Identified in the serum of Mrs. Kelleher
Zinc Endopeptidases
- All Kell Antigens can ONLY BE FOUND IN RBCs
- Encoded on Chromosome 7 Kell Antigens (K/k, Kpa/Kpb and Jsa/Jsb)
o KEL Gene = 19 Coding Exons - Have 15 Cysteine Amino Acids which forms
o Mutations in Kell Gene = K0 or Kell Null Disulfide Bonds
o Makes them Resistant to Enzymes but
Antigens:
Very Sensitive to Sulfhydryl Reagents
1. K and k
- Examples of Sulfhydryl Reagents = 2-
o Antithetical Antigens
Mercaptoethanol, Dithriothreitol, 2-
o K = Kell = Low Prevalence Antigen
Aminoethylisothiouronium Bromide (2-AET)
(Less than 9% of Population)
and ZZAP
o k = Cellano = High Prevalence
Antigen (More than 90% of Kx Antigen
Population) - Spans the RBC Membrane 10x and is an RBC
o Well developed at birth: Transport Protein
▪ K = 10 Weeks of Gestation - Kx Antigen is linked to Kell Glycoprotein via
▪ k = 7 Weeks of Gestation a Single Disulfide Bond = Forms Km Antigen
o K Antigen is VERY IMMUNOGENIC and - Resistant to Sulfhydryl Reagents
is SECOND ONLY TO D ANTIGEN in
stimulating antibody production. ***The Kx Antigen is always present on RBC
2. Kpa and Kpb Membranes but sometimes cannot be detected or
o Kpa = Low Prevalence Antigen (2% of are only in small amounts because the Disulfide
Whites) = Results to a Reduced Bonds of Kell Antigens form a cloud which masks
Amount of Kell Glycoprotein in RBC detection of the Kx Antigen and appear decreased
Membrane in number.
o Kpb = High Prevalence Antigen ***If Kell Antigens are not present, there is no cloud
(99.9% of Population) to cover Kx Antigens making their detection easier
o Kpc = Very Rare and appear increased in number.
o Named as Kp since first found in Mrs.
Penney Anti-K
3. Jsa and Jsb - IgG that reacts well at AHG Phase and may
o Jsa = Low Prevalence Antigen (20% of also react at Immediate Spin Phase
Blacks and 0.1% of Whites) - Produced via Exposure through Pregnancy
o Jsb = High Prevalence Antigen and Transfusions
o Named as Js since first found in Mr. - Anti-K is the 3rd Most Common Antibody Seen
John Sutter in Blood Bank
4. Kx Antigen (ISBT 019) o 1st is ABO Antibodies
o Encoded on X Chromosome o 2nd is Rh Antibodies
(Chromosome 23 or Sex - IgM Anti-K = Very Rare = Associated w/
Chromosome) Bacterial Infections (E. coli 0125:B15) which
o XK1 Gene was said to have a Somatic K-Like Antigen
o Not Part of the Kell Blood Group o It is interesting to note that the IgM
System but is Closely Associated to Anti-K disappeared after recovery of
Kell Antigens the patient.
o Found in RBCs and Other Tissues such - Most Reliable Method of Detecting Anti-K =
as Brain, Lymphoid Organs, Heart and INDIRECT ANTIGLOBULIN TEST
Skeletal Muscle - Polyethylene Glycol = Increases Reactivity of
o Present in All Individuals Anti-K
45
- RBC Destruction is usually Extravascular via - Caused by Several Mutations and Deletions
Splenic Macrophages at the XK1 Gene leading to Absence of Kx
- Causes Severe HTR and Severe HDFN w/ Fetal Antigen and Km Antigen
Anemia - Results to Marked Depression of All Other Kell
Antigens
Anti-Kpa and Anti-Jsa
o Denoted as w for weak: K-k+w Kp(a-
- Rare since they are antibodies to Low
b+w)
Prevalence Antigens
- X-Linked Mode of Inheritance through Carrier
- Most Often Detected through Unexpected
Mothers
Incompatible Crossmatches or Cases of
o Very Rare and is Only Manifested in
HDFN
Males
- Similar to Anti-K in Clinical Significance
- Since Kx Antigen is a Membrane Transport
- Original Anti-Kpa = Naturally Occurring
Protein, its absence causes Significant RBC
- Most Anti-Kpa and Anti-Jsa = Results from
Membrance Abnormalities
Transfusion or Pregnancy
- McLeod Syndrome:
Anti-k, Anti-Kpb and Anti-Jsb o Acanthocytosis (RBCs w/ Irregular
- Very Rare since Only a Few People Lacks Shapes and Protrusions)
these Antigens o Decreased RBC Deformability and
- Parallels Anti-K in Clinical Significance Reduced RBC Survival
- Tested in Blood Banks using DTT or AET-treated o Chronic Hemolytic Anemia w/
RBCs Reticulocytosis, Bilirubinemia,
o If Reactivity is Abolished = Suggests Splenomegaly and Reduced
Antibody Related to Kell System Haptoglobin
o To Confirm = Test with Antigen- o Progressive Muscular Dystrophy
Negative RBCs between ages 40-50
o DTT also abolishes reactivity of JMH o Cardiomegaly leading to
and High Prevalence Antigens of LW, Cardiomyopathy
Lutheran, Dombrock, Cromer and o Areflexia = Lack of Deep Tendon
Knops BGS Reflex
o Choreiform Movement = Well
K0 Phenotype or Kell Null Coordinated but Involuntary
- Lacks expression of all Kell Antigens Movements
- K0 Individuals have No RBC Membrane o Elevated CK-MM and Carbonic
Abnormality and Survive Normally in Anhydrase Levels
Circulation - Found to be Associated with Chronic
- K0 Individuals still have the Kx Antigen as it is Granulomatous Disease
not encoded on the same chromosome as o CYBB Gene = Found in the X
Kell Antigens Chromosome and Close to XK1 Gene
- Very Rare o Any deletion to XK1 Gene could also
- Produces an Antibody called Anti-Ku or Anti- result to deletion in the CYBB Gene
K5 causing Both McLeod Phenotype
o Recognizes Ku (Kell Universal) and Chronic Granulomatous Disease
Antigen Present on All RBCs except K0 o Not all males w/ McLeod have CGD,
Individuals nor do all males w/ CGD have
o Of Single Specificity and Cannot be McLeod
Separated into Components o CGD = Deletion of CYBB Gene =
o Causes both HDFN and HTR Absence of NADPH Oxidase =
- K0 Individuals may only receive Kell Negative Inability of Phagocytes to Kill Aerobic
Blood Bacteria
o WBCs can Phagocytize Bacteria but
Cannot Kill Them
McLeod Phenotype and McLeod Syndrome - McLeod Males w/ CGD produces Anti-
- Named after McLeod who appeared to be Kx+Km
K0 but Demonstrated Weak k, Kpb and Jsb o Reacts Strongly w/ K0 RBCs
Expression via Adorption-Elution Methods o Reacts w/ Normal Kell Phenotypes
but Weaker
o No Reaction w/ McLeod RBCs
46
- McLeod Males w/o CGD produces Anti-Km - Have been identified in other body tissues
(Brain, Colon, Endothelium, Lung, Spleen,
Altered or Weakened Expression of Kell Antigens
Thyroid, Thymus and Kidney Cells)
- Termed as Kmod = Very Weak Kell Expression
- Destroyed by Ficin, Papain, Bromelin,
= Requires Adsorption-Elution Tests to Detect
Chymotrypsin and ZZAP
- Appears as having Enhanced Kx Expression
- Resistant to DTT, AET and Glycine-Acid EDTA
- Causes of Weak Kell Antigens:
- Neuraminidase and Purified Trypsin =
o McLeod Phenotype
Reduces Molecular Weight of Fya and Fyb
o Suppression of Kpa Gene due to Cis-
but Does not Destroy Antigenic Activity
Modified Effect
o Gerbich Negative Phenotypes (Ge: - Biochemistry of Duffy Antigens:
2, -3, 4 and Ge: -2, -3, -4) - Duffy Glycoprotein = Protein where Duffy
▪ Relationship w/ Kell is not Antigens are Located
understood o Member of the Chemokine Receptor
- Produces an antibody that resembles Anti- Superfamily
Ku but is different o AKA Duffy Antigen Receptor for
Chemokines (DARC)
***K- Negative RBCs appear to acquire Kell Antigens
o Traverses RBC Membrane 7x and has
as there was a case wherein a K- Patient acquired a
2 Predicted Disulfide Bridges
K-Like Antigen during Streptococcus (Enterococcus)
o 42nd Amino Acid differentiates Fya
faecium infection.
and Fyb:
***Autoantibodies to K, Kpb and K13 have also been
▪ Fya = Glycine
reported.
▪ Fyb = Aspartic Acid
Kell
Phenotyp o Fy3 = Located on the 3rd Extracellular
Antigen Km Kx Antibody
e Loop
s
k, Kpb, Norm Alloantibod o Fy6 = Involves Amino Acids 19-25
Common Weak o Acts as a Receptor for Plasmodium
Jsb… al y
Increase Anti- vivax and Able to Bind Variety of
K0 None None
d Ku/Anti-K5 Proinflammatory Cytokines
Anti-Kx+Km
Trace k, Fy Phenotype or Fy (a-b-):
(CGD)
McLeod Kpb, None None - Silent Allele of Duffy BGS
Anti-Km
Jsb…
(Non-CGD) - Divided into Fy (a-b-) Blacks and Fy (a-b-)
End of Kell BGS Whites
- Fy (a-b-) Blacks is actually an Fyb Variant w/
Duffy Blood Group System (ISBT 008)
a Change in the Promoter Region of the Gene
- Named after Mr. Duffy who was a Multiply
o Results to Disruption of Binding Site for
Transfused Hemophiliac
mRNA Transcription in RBC
- Encoded on Chromosome 1
o Leads to Fy (a-b-)
o Both Fy and Rh are found on
o Individuals w/ this phenotype DO NOT
Chromosome 1
EXPRESS Fyb on RBCs but EXPRESSES
o Fy Locus is Syntenic to the Rh locus
Fyb in other Tissues
▪ Syntenic means that they are
o Since Fyb is still found in other tissues,
found on the same
Anti-Fyb is NOT produced
chromosome but are far
- Fy (a-b-) Whites = No Duffy Protein on RBCs or
enough apart that they
Tissues = Forms Anti-Fyb and Anti-Fy3
segregate independently
- Theorized as an Evolutionary Adaptation as
- 3 Common Alleles:
Fy (a-b-) is the most common phenotype
o Fya = Codes for Fya Antigen
found in Africa which is endemic of Malaria.
o Fyb = Codes for Fyb Antigen
o Fya and Fyb Antigens are receptors of
o Fy = Silent Allele = Major Allele in
Plasmodium vivax
Blacks
o Africans developed the Fy (a-b-) to
Fya and Fyb Antigens: make them more resistant to Malarial
- Identified on Fetal RBCs as early as 6 Weeks Infections
of Gestation o Fy (a-b-) RBCs also resist infection IN
- Well Developed upon birth VITRO by Plasmodium knowlesi
- Not found on Platelets nor WBCs (monkey malarial organism but also
causes malaria in humans)
47
Occurrence of Duffy Phenotypes: ▪Produced most often by Fy (a-

1. Fy (a+b-) = More Common in Whites; Most b-) Whites found in Whites,
Common in Chinese Cree Indian Families and
2. Fy (a-b+) = More Common in Whites Rarely Blacks
3. Fy (a+b+) = More Common in Whites; Rare in 3. Fy5 Antigen and Antibody
Blacks o Appears to be a Result of Interaction
4. Fy (a-b-) = Most Common in Blacks; Very between Rh Complex and Duffy
Rare in Whites Glycoprotein
o Fy (a-b-) Individuals or Rh Null
Anti-Fya and Anti-Fyb:
Individuals DO NOT PRODUCE Fy5
- Anti-Fya = 3x Less Common than Anti-K
Antigen and are at Risk of Producing
- Anti-Fyb = 20x Less Common than Anti-Fya
Anti-Fy5
- IgG Antibodies that react best at AHG Phase
o Fy5 is not destroyed by enzymes
- Enhanced by LISS
o Anti-Fy5
- Rarely binds to complement
▪ Reacts w/ All Fy (a+) or Fy (b+)
- Do not React w/ Enzyme Treated RBCs
Rh+ RBCs
- Show Dosage Effect
▪ Does Not React w/ Fy(a-b-)
o Homozygous = Fy (a+b-) and Fy (a-
RBC but Reacts w/ Fy(a-b-)
b+) = Greater Reaction
Fy: -3 Phenotype suggesting
o Heterozygous = Fy (a+b+) = Weaker
that it is Different from Anti-Fy3
Reaction
▪ Does not React w/ Fy (a+) or
o FyaFya Whites = Double Dose of Fya
Fy (b+) Rh Null RBCs
o FyaFy Blacks = Only Single Dose of Fy
▪ Only Weakly Reacts w/ Fy
- Associated w/ Acute and Delayed HTR and
(a+) or Fy (b+) D- RBCs
HDFN
▪ Serum containing Anti-Fy5
- Must be given Fy (a-) or Fy (b-) Blood
also contain Anti-Fya
Other Duffy Antigens:
Kidd Blood Group System (ISBT 009)
1. Fyx
- Simple and Straightforward System w/ Only 3
o Does not produce a distinct antigen
Antigens
but rather an Inherited Weak Form of
- Discovered in Mrs. Kidd whose infant had
Fyb
HDFN
o Due to a Reduced Amount of Duffy
- Encoded on Chromosome 18 by SLC14A1
Glycoprotein on RBC Surface
Gene
o May react w/ Anti-Fyb
o SLC14A1 = Solute Carrier Family 14
o Found in White Population
Member 1
o Individuals w/ Fyx may type Fyb (b-)
o Urea Transporter Gene w/ 11 Exons
but their RBCs are capable of
o Jka and Jkb differs w/ an Amino Acid
adsorbing and eluting Anti-Fyb
Substitution at 280th Position located
suggesting presence of Weak Fyb
at the 4th Extracellular Loop of the
o Have Depressed Expression of Fy3
Kidd Glycoprotein
and Fy5 Antigens
o No Anti-Fyx is Produced Jka and Jkb Antigens
2. Fy3 Antigen and Antibody - Found on RBCs of Most Individuals
o Universal Antigen or Precursor - Well Developed on Fetal RBCs
common to both Fya and Fyb - Jka = Detected at 11 Weeks of Gestation
o Not Destroyed by Enzymes - Jkb = Detected at 7 Weeks of Gestation
o Absence of the Antigen in Fy (a-b-) - Codominant Alleles
Whites who lack the Duffy - Not very immunogenic
Glycoprotein produces Anti-Fy3 - Enzymes enhances Reactivity w/ Kidd
o Fy (a-b-) Blacks = Rarely Produces Antibodies
Anti-Fy3 - Not Affected by Chloroquine, DTT, AET or
▪ Blacks who produce Anti-Fy3 Glycine-Acid EDTA
also produces Anti-Fya - Not Found on Platelets nor WBCs
o Anti-Fy3
Phenotype Whites Blacks Asians
▪ Reacts to All RBCs except
Jk (a+b-) 28 57 23
those w/ Fy (a-b-) Phenotype
▪ Inseparable Anti-FyaFyb Jk (a-b+) 23 9 27
48
Jk (a+b+) 49 34 50 o Studies on 2 Individuals showed a

Very Very 0.9 in Marked Defect in Urine
Jk (a-b-)
Rare Rare Polynesians Concentration
- Results from Inheritance of the Silent Jk Allele
Anti-Jka and Anti-Jkb - Produces Anti-Jka, Anti-Jkb and Anti-Jk3
- Notorious Reputation - Anti-Jk3
o Shows Dosage o Jk3 Antigen = Universal Antigen and
▪ Reacts Strongly w/ Jk (a+b-) or Common Precursor of Jka and Jkb
Jk (a-b+) but may not react o IgG Type that reacts at AHG Phase
w/ Jk (a+b+) o Inseparable Anti-JkaJkb
o Often Weak o Determined by Antigen Typing since
▪ Must be Enhanced w/ Use of only produced by Jk (a-b-)
LISS or Polyethylene Glycol Individuals
w/c Promotes IgG o Reactivity is enhanced by Enzymes
Attachment o Severe Immediate and Delayed HTRs
o Found in Combination w/ Other and Mild HDFN
Antibodies o Found in Far East and Pacific Islanders
o Binds to Complement
▪ Use Polyspecific Anti-IgG and
- In(Jk) = Inhibitor Jk Gene
Anti-Complement to Detect
o Dominant Pattern of Inheritance
o All of these Factors make them
o Found in a Japanese Family
difficult to detect
o Individuals w/ this gene are termed as
- Anti-Jka is More Frequently Encountered than
DOMINANT TYPE Jk (a-b-)
Anti-Jkb but neither antibodies are common
▪ RBCs w/ Dominant Type Jk (a-
- IgG Type that is Reactive at AHG Phase
b-) adsorb and elute Anti-Jka,
- May also be Partly IgM
Anti-Jkb and Anti-Jk3
- Made in response to Pregnancy or
indicating WEAK EXPRESSION
Transfusion
OF Jk ANTIGENS
- Titer of Anti-Jka and Anti-Jkb declines rapidly
▪ DO NOT PRODUCE ANTI-Jk3
in vivo
o In(Jk) Gene does not reside at the Jk
o Strong Antibody following a
Locus and Genetic and Molecular
Transfusion Reaction may be
Basis is still unknown
Undetectable in a Few Weeks or
Months ***Autoantibodies w/ Kidd Specificity (Anti-Jka, Anti-
- COMMON CAUSE OF DELAYED HTRs Jkb and Anti-Jk3) are rare but have been associated
o Most often Extravascular but with AIHA.
sometimes Intravascular END OF KIDD BGS
- Rarely Associated w/ HDFN Lutheran Blood Group System (ISBT 005)
- Discovered on a patient who had Systemic
Lupus Erythematosus and was donated with
Jk (a-b-) Phenotype and Recessive Jk blood by Mr. Lutteran but his blood sample
- Lacks Jka, Jkb and Jk3 Antigens was incorrectly labelled
- Abundant among Polynesians - Composed of 20 Antigens numbered
- Jk (a-b-) RBCs resist lysis in 2M Urea Solution through Lu22
o Urea crosses RBC Membrane = o Lu10 and Lu15 = Obsolete
Causes Osmotic Imbalance = Influx of o 4 Pairs of Antithetical Antigens
Water = Lysis of Normal Cells o Mostly High Prevalence Antigens
o Jk (a+) and Jk (b+) RBCs = Lysis in 2M - Encoded on Chromosome 19
Urea w/in 1 Minute - Linkage between Lu and Se Gene (FUT2) = 1st
o Jk (a-b-) RBCs = Lysis in 2M Urea Example of Autosomal Linkage Described in
Delayed by 30 Minutes Humans
o Used to Screen Population for the Null Lua and Lub:
Jk Phenotype - Codominant Alleles
- No Clinical Abnormalities have been - Most Individuals are Lu (b+)
associated - Only 8% of Whites and 5% of Blacks are Lu
- Most have Normal BUN, Creatinine and (a+)
Serum Electrolytes
49
- Varies from Individual to Individual Anti-Lub

- Detected on Fetal RBCs at 10-12 Weeks of - Primarily IgG but IgM and IgA Types have also
Gestation but POORLY EXPRESSED AT BIRTH been found
o As a result, HDFN is very rare and only - Reactive at 37C @ AHG Phase = Warm
mild. Alloantibody
- EXPRESSED FULLY BY AGE 15 - Produced through Pregnancy and
- Not Detected on Platelets nor WBCs Transfusions
- Lutheran Glycoprotein is widely distributed in - Reacts weaker w/ Lu (a+b+) RBCs
tissues (Brain, Lung, Pancreas, Placenta, - Implicated w/ Shortened Survival of
Skeletal Muscle and Hepatocytes) Transfused RBCs and Post-Transfusion
o Found Especially in Fetal Hepatic Jaundice
Epithelial Cells - Associated w/ HTR and only rarely HDFN
o Presence of Lutheran Glycoprotein on
Lu (a-b-) Phenotypes
Placental Tissue = Results in
- 3 Types:
Absorption of Maternal Antibodies to
o Dominant Type Lu (a-b-)
Lutheran Antigens = Decreases
o Recessive Type Lu (a-b-)
Likelihood of HDFN
o Recessive X-Linked Inhibitor Type
- Resistant to Ficin, Papain and Glycine-Acid
- Dominant Type Lu (a-b-)
EDTA
o Individuals w/ Dominant Type Lu (a-
- Destroyed by Trypsin and Chymotrypsin
b-) may still pass Normal Lutheran
Phenotype Percent Population Genes to their offspring
Lu (a+b-) 0.15 o Linked to a Dominant Regulator
Lu (a-b+) 92.35 Gene called In(Lu) for Inhibitor
Lu (a+b+) 7.5 Lutheran Gene
Lu (a-b-) Very Rare ▪ In(Lu) Phenotype =
***Lu (a-b+) is the most common Lutheran Associated w/ Mutations in
Phenotype. the Erythroid Kruppel-Like
Factor (EKLF)
Biochemistry of Lutheran Antigens: ▪ In(Lu) Phenotype = Caused
- Lutheran Antigens = Located on Type 1 by Inheritance of a Loss-of-
Transmembrane Protein Function Mutation on 1 Allele
o Transmembrane Protein has 2 Forms of EKLF
due to Alternative RNA Splicing: o Carry trace amounts of Lutheran
o Longer Lu Glycoprotein Antigens as demonstrated by
o Shorter Basal Cell Adhesion Molecule Adsorption-Elution Studies
(B-CAM) ▪ Persons w/ 2 Normal Lub
- Lutheran Proteins = Multifunctional Adhesion Genes who have Dominant
Molecules = Binds Laminin = Implicated in In(Lu) will still type as Lu (a-b-)
Sickle Cell Disease but will adsorb and elute Anti-
Anti-Lua Lub.
- IgM Naturally Occurring Saline Agglutinins o Do not Produce Anti-Lu3
that React Better at Room Temp than 37C o In(Lu) also Reduces Expression of
- Few Anti-Lua reacts at 37C via Indirect CD44 and Induces Weak Expression
Antiglobulin Test of P1 and i Antigens
- Capable of Binding Complement - Recessive Type Lu (a-b-)
- Encountered as an Incompatible o Caused by 2 Rare Silent Alleles LuLu
Crossmatch or During Antibody Workup for at the Gene Locus
Another Specificity o Classic Null Lutheran Phenotype
- Has Characteristic Loose, Mixed-Field o Truly Lack all Lutheran Antigens
Reactivity in Test Tube o Produces an Inseparable Anti-Luab
- Clinically Insignificant and No Documented called Anti-Lu3
Cases of Immediate HTRs o Anti-Lu3
- Rare and Mild Delayed HTRs may sometimes ▪ Recognizes a
occur Common/Universal Antigen
Lu3 that is present whenever
Lua and Lub are present
▪ AHG Reactive
50
Made only by Individuals of

▪ • Ag: developed @ birth but are more weakly
Recessive Lu (a-b-) on cord RBCs than on adult RBC
Phenotype • Absent from people w/ PNH III
o Have Normal Expression of P1 and i
XG SYSTEM
Antigens • Symbol: XG
o Recessive Lu (a-b-) results to a • 1962, Anti-Xga discovered in serum of
Truncated Glycoprotein that Would multiple transfused man
Not be Incorporated in the RBC • Ab detected an antigen w/ higher
Surface Membrane prevalence in females than in males
- Recessive X-Linked Inhibitor Type • Ag named after X chromosome & g for
“Grand Rapids”, where px was treated
o Found in a Large Australian Family
• CD99 aka MIC2 & 12E7
o Only Manifested in Males • CD99 & Xga, their genes are adjacent and
o Carried Trace Amounts of Lub as homologous kaya in the same system
detected by adsorption-elution • !!Directly proportional ang Antigens
studies • ^but Xg(a-) in males, 68% have high CD99
o Caused by Inheritance of XS2 Gene = and 32% have low
X-Linked Inhibitor of Lutheran • Xga and CD99 escape X chrom inactivation
• 2 CD99- Japanese peeps have alloanti-
▪ Suppresses in a Hemizygous
CD99
State = Only 1 Copy of the
Allele is Needed = All Genes in SCIANNA SYSTEM
X Chromosome are • Symbol: SC
Hemizygous • Named after 1st Ab marker
• 1962, new high prevalence Ag named Sm
Other • 1963, low prevalence Ag Bua
Mode of Gene RBC Anti-Lu3 • ERMAP = RBC adhesion protein
Lu
Inheritanc Responsibl Antigens Productio
Antigens
e e Affecte n
DOMBROCK SYSTEM
d
• Symbol: DO
Extremel P1, i,
Dominant EKLF
y Weak CD44
None • 1965, 1st antibody maker, Mrs. Dombock
Totally
• 1973, antithetical Ag identified
Recessive Lu None Yes • Gy (a-) = Hy-
Absent
Extremel • But in blacks, Hy- are weakly Gy(a+)
X-Linked XS2 None None • Mono-ADP ribosyltransferase 4 (mono-ART4)
y Weak
• Mono-ART4 is attached to RBC membrane
by a GPI anchor
OTHER MINOR BLOOD GROUPS:
• Doa and Dob are poor immunogens
COLTON SYSTEM
DIEGO SYSTEM
• Symbol: CO
• Symbol: DI
• 1967, 1st Ab marker
• Marked after the 1st Ab marker on a
• Should have been named Calton, but
Venezuelan fam during an investigation of
handwriting on tube was misread
HDFN
• Co3, present on all RBCs except those of
• 1955, Anti-Dia had caused HDFN in a
very rare Co(a-b-) phenotype
Venezuelan baby
• Co4, identified on RBCs from 2 individuals w/
• 1957, Anti-Dib described
Co(a-b-) phenotype
• Ag: expressed on RBC of Newborn
• Aquaporin 1, accounts for 80% of water
• Wrb req presence of both GPA
readsorption in kidneys
(glychophorin A, MNS system) of MNS blood
• Anti-Coa cause HTR & HDFN
group system
• Anti-Cob cause HTR & mild HDFN
• !!Bpa – sensi to papain
• Anti-Co3 reacts w/ all Co(a+) & Co(b+)
• Autoanti-Wrb in serum of Warm AIHA px
RBCs, cause severe HDFN
• Anti-Dia, Anti-Dab and Anti-Wra are clinically
LANDSTEINER-WIENER SYSTEM
significant, cause severe HTR and HFDN
• Symbol: LW
• Anti-Wra is a relatively common Ab
• Origin along w/ discovery of D Ag of the Rh
BGS
YT SYSTEM
• 1940, Landsteiner & iener reported Ab
• 1956, first antibody marker
produced in Rabbits after injection w/ RBCs
• Used “t” in px last name, Cartwright
• LWab was originally defined by Ab made by
• “Why T” became “Yt”
indiv w/ an inherited LW(a-b-) phenotype
• Rare phen: Yt (a-b+)
• LW5 = LWa
• Ag: Antithetical, represent AA substitution on
• LW6 = LWb
GPI-linked RBC AChE
• LW7 = LWab
• Mrs. Big, made anti-LWab
51
• !!RHnull RBCs also type LW(a-b-), considered CROMER SYSTEM

to be the only true LW- RBCs bc they fail to • 1965, Ab was found in black px, Mrs.
elicit formation of anti-LW in animals Cromer, reacted w/ all RBCs except her
• Injection of Mrs. Big’s LW(a-b-) RBCs into own & 2 siblings
guinea pigs caused formation of anti-LW • Originally thought her Ab recognized an Ag
• Phenotypes w/o agg: LW (a-b-) Big, LW(a- antithetical to Goa of the Rh BGS
b-) Rhnull • Antibody named Anti-Cra in 1975
• There is a phenotypic relationship between • DAF, complement regulatory protein
the D Ag & Anti-LW • Glycoprotein encoded by the gene is
• Anti-LW usually reacts strongly w/ D+ attached to the RBC membrane thru a GPI
• Weak anti-LW may react only w/ D+ RBCs linkage
• More LW Ag sites on D+ RBCs from adults • Cr(a-) phenotype is typically found in blacks
than on D- RBCs & is not found in whites
• Anti-LW reacts equally well w/ cord RBCs • Tca, Tcb, Tcc are antithetical
regardless of their D type • Dr(a-) RBCs have weakened expression of
• Distinguishing Anti-LW from anti-D is most all other high-freq Crom Ag due to markedly
easily accomplished by testing DTT treated reduced copy of DAF
D+ RBCs: D Ag not denatured so anti-D • α-Chymotrypsin is used to distinguish
would be detected; LW Ag is resistant to specificities in system from other blood
RBCs treated w/ enzymes & glycine-acid group Ab
EDTA
• LW glycoprotein is part of band 3/Rh KNOPS SYSTEM
macrocomplex • CR1 – complement receptor 1
• Rhnull lacks LW glycoprotein • Named after Mrs. Knops, 1st Ab marker
• LW Ags depressed during pregnancy & • Antithetical pairs Kna & Knb, McCa & McCb,
some diseases like lymphoma & leukemia S1a & Vil
• AutoAbs by these px may appear to be • Antigens weaken upon storage of adult
AlloAb RBCs
• Autoanti-LW is common in serum of warm • Serologically, Ag have been grouped
AIHA px together because their corresponding Ab
demonstrate variable rxn
CHIDO/ROGERS SYSTEM • Not neutralized by pooled normal serum
• Symbol: CH/RG • Difficult to adsorb & elute
• Named after 2 Ab producers, Ch for Chido • Anti-Kna is frequently found in multiply
& Rg for Rodgers transfused individuals & multispecific sera
• 1967, Ch • Anti-S1a is more freq found in blacks
• 1976, Rg • CR1: binds C3b & C4b & processes immune
• Serologically, both characterized as complexes for transpo to the liver & spleen &
nebulous bc Ag strength on diff samples of subseq clearance from circulation
RBCs was variable • Also acts as receptor for several pathogenic
• Both Antibodies can be neutralized by organisms
plasma
• Ch & Rg Ags not intrinsic to RBC membrane INDIAN SYSTEM
• Found on C4 (check table) • Symbol: IN
• Neutralization of anti-CH & anti-Rg w/ • Named bc 1st In(a+) indiv were from India
pooled plasms is often used as part of the ID • CD44 is an adhesion moleculeIn(a-b-) was
of these Ab in a px’s serum found in only 1 individual w/ CDA whose
RBCs also typed Co(a-b-)
GERBICH SYSTEM • Decreased RBC survival w/ anti-Ina
• 1960, Mrs. Gerbich the 1st Ab producer • Immediate HTR due to anti-Inb
• GPC & GPD help maintain RBC membrane
integrity thru interaction w/ Protein band 4.1 OK SYSTEM
• Ge2 & Ge4 are ficin & papain sensitive • Symbol: OK
• Ge3 is ficin resistant • 1979, Anti-Oka identified & named after 1st
• In HDFN by anti-Ge3, severe anemia was Ab marker Mrs. Kobutso
late onset, after birth, associated w/ • Bc Ko was already in use, 1st 2 letters were
inhibition of erythroid cell growth, in 1 case switched to Ok
there was also early onset of hemolysis • Her parents were cousins from a small Jap
• Anti-Ge2, most common Ab, produced by island (omggg) & two of three siblings were
Ge:-2,3,4 phenotype … more common in also Ok(a-)
peeps by Ge:-2,-3,4 phenotype and Ge:-2,- • RBCs from 400 indiv from same island were
3,-4 phenotypes Ok(a+)
• Anti-Ge3 less frequent, made by by Ge:-2,- • 2 additional Ags: OKGV, OKVM
3,4 phenotype and Ge:-2,-3,-4 phenotypes • CD147 – member of immunoglobulin
• Anti-Ge4 ver rare Ab superfamily that mainly functions as
receptors & adhesion molecules
52
• One other Ab was found w/c caused • Strength of Sda on adult RBCs varies & is
reduced survival of 51Cr-labeled Ok(a+) markedly reduced in pregnancy
RBCs injected into original Ab marker • Anti-Sda can naturally occur in sera of
individuals who are Sd(a-)
RAPH SYSTEM • Anti-Sda is an IgM agglutinin reactive @ RT,
• Ag was originally defined by 2 monoclonal but it can be detected in IAT & doesn’t
Abs; it has since been recognized by human react w/ cord RBCs
polyclonal Abs • Neutralization of refractile agglutinates by
• Name derived from monoclonal, & Eleanor urine is a technique used to ID anti-Sda bc
Roosevelt, the lav where Ab was produced soluble Ags is present in urine
• Named Raph for the 1st px to make the
alloanti-MER2 **Review:
• 3 other people w/ alloanti-3 had end-stage Systems that Produce Cold Antibodies = IgM Type =
renal disease LIPMAN
• CD151 – a tretraspanin w/c appears to be - Anti-Lea, Anti-Leb and Anti-Lua
essential for the assembly of BM in kidneys & - Anti-I, Anti-i
skin - Anti-P1
• MER2 expression decreases over time w/ - Anti-M
increasing maturation of erythroid cells - Anti-A, Anti-B, Anti-H
• Could be MER2- (RBC: (-); others: (+)) - Anti-N
• True MER2-: have alloanti-MER2
• Sensi to: Trypsin, α-chymotrypsin, pronase & Systems that Produce Warm Antibodies = IgG Type
AET - Rh
- Kell (All Kell Antibodies)
JMH SYSTEM - Duffy (All Duffy Antibodies)
• Symbol: JMH - Kidd (All Kidd Antibodies)
• 1978, large # of samoles w/ this Ab were - S, s
named for 1st Ab marker John Milton Hagen - Anti-Lub
• Anti-JMH especially seen in px 50 y.o. and - Autoanti-P
above
• JMH protein is the GPI-linked glycoprotein Enhanced by Enzymes:
CD108 - Kidd
• JMH1 represents Ag recognized by Abs - Rh
made by individuals w/o JMH protein - Lewis
• Most Anti-JMH are not foundin px who lack - I
JMH protein or who have one of the variant - P
of JMH phenotypes
• JMH- status is acquired and can be transient Destroyed by Enzymes = Fya and Fyb (Duffy) and MNSs
• JMH levels decline during later years of life, Destroyed by Sulfyhydryl Reagents = Kell Antigens
sometimes to the point of not being Systems that Exhibit Dosage:
serologically detected - Rh except Anti-D
• Once reduced, can produce anti-JMH - Kidd
• Autoanti-JMH w/ DAT (+) result has never - Duffy
been associated w/ autoimmune RBC - MNSs
destruction
• Rare example of alloanti-JMH in individuals Clinically Significant Antibodies:
whose RBCs express variant forms of CD108 - ABO Antibodies
may be clinically significant - Anti-Rh
- Anti-PP1Pk, Alloanti-P, Autoanti-P
GILL SYSTEM - Pathologic Autoanti-I, Autoanti-i
• Symbol: GIL - Anti-S
• 1980, Anti-GIL 1st identified - Anti-s
• 2002, Ag was not raised to system status until - Anti-U
this year when it was shown that GIL is - Lewis Antibodies
genetically discrete from all other BGS - Anti-K, Anti-Kpa and Anti-Jsa
- Duffy, Kidd and Lutheran Antibodies (Except Anti-
Sda Lua)
• Named for Sid, head of the maintenance
dept @ Lister Institute in London Clinically Insignificant Antibodies:
• His RBCs had been used for many years as a - Anti-LW
panel donor, reacted strongly w/ examples - Anti-P1
of a new Ab - Alloanti-I
• Soluble form of Sda is Tamm-Hrosfall - Anti-M
glycoprotein in urine - Anti-N
• Ag is not expressed newborns, but is in their - Anti-k, Anti-Kpb and Anti-Jsb
saliva, urine & meconium - Anti-Lua
53

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LECTURE NOTES BY: JOASH-EVANGEL Z.

CAMPOS, RMT, MLS (ASCPi)

HISTORY OF TRANSFUSION MEDICINE ▪ “And he said, What hast thou

18th Century: - Milk was advocated as a potentially

- 1939: Levine and Stetson o Recommended sodium bicarbonate

(6) Plastic Blood Bags

(7) Component Therapy

CHANGES OVER TIME

Naïve/Virgin Cells (Th0, Tc0)

**Adaptive is more powerful due to specificity &

**Douglas & Wright

Classical Pathway (specific)

o Properdin: stabilizes C5 convertase

** HLA-DMA and -DMB genes are both required

RBC Antigen Compositions:

**About 600 molecules (probability for 2 IgGs to

Immediate Spin Antiglobulin Phase /

Exception to the Law of Independent Assortment

Linkage & Haplotypes:

Applications of Molecular Testing in BB:

US Food & Drug Administration (FDA) - Discovery of the test:

Monoclonal AHG Production

ANTIBODIES CAPABLE OF BINDING COMPLEMENT

DIRECT ANTIGLOBULIN TEST (DAT) PANEL

DAT Panel: Patterns of Reactivity in AIHA

Confirm Negative Results:

APPLICATION TESTS IN VITRO SENSITIZATION

2. Reaction medium - All negative antiglobulin test reactions must

Antibody Potentiators and Lectins:

→ Solid Phase Technology

POTENTIATOR DESCRIPTION ADVANTAGE DISADVANTAGE

ABO BLOOD GROUP SYSTEM (ISBT NO. 001)

ABO Blood System

Formation of the O Antigen

o Enzyme modifies the type 1 precursor o Ulex eurpaeus

- Infants appear as A2 at birth due to

Gradient of the subgroups of A: # of A antigen sites

The Inheritance of the Rare h Allele:

- Rare phenotypes in which RBCs are o ‘hangovers’ in Type A blood group

1. Obtain patient’s data (96 years old patient)

▪ Or by adding 1 or 2 drops more 1+ 4+ 4+ 0

1. Excess amount of blood group-specific solube

patient receives O cells)

o Plasma expanders such as dextran & present on the person’s red

Resolution in the BB Lab:

→ Has weak anti-B which reacts to ordinary B Gene products: Glycosyltransferases

TESTING THE SECRETOR STATUS USING SALIVA

**AABB: Add in lighter reagents before dark colored Contents:

Grade the results

Rh BLOOD GROUP SYSTEM

1. Based on genetic inheritance:

A. FISHER-RACE: DCE Terminology

Humans can form anti-Rh antibodies & anti-LW

o if h precedes the r = refers to either

C. ROSENFIELD: Alphanumeric Terminology

ANTIGEN NUMERIC ISBT NUMBER

Weak D Variations of D antigen expression

Patient can be safely given Rh-positive blood (+)

c. Partial D (D Mosaic) A. Rhnull

**Individuals regardless of type are negative for