IMMUNOHEMATOLOGY, LEC
Prof. Ruby Garcia-Meim, RMT, MSPH
UNIT I: HISTORICAL BACKGROUND OF
BLOOD BANKING
• In 1492
o blood was taken from 3 young men
and given to the stricken Pope
Innocent VII in the hope of curing
him; unfortunately, all four died
• 1869: Braxton Hicks
o recommended sodium phosphate
• 1901: Karl Landsteiner
o in discovered the ABO blood
groups
-> Nobel Prize
-> Safer transfusion
-> collected blood from himself and 5
associate. Separated the serum from
red cell
o A ag – “A” Anti B
o B ag – “B” Anti A
o A, B ag – “AB” No Ab
o No Ag – “O” Anti AB
• Edward E. Lindemman
o carried out vein-to-vein transfusion
of blood by using multiple syringes
and a special cannula for puncturing
the vein through the skin
- requires assistance
• Unger
o designed his syringe-valve
apparatus that transfusions from
donor to patient by an unassisted
physician became practical
- Does not require many assistance
• 1914: Hustin
reported the use of sodium citrate as an
anticoagulant solution for
transfusions
- Acidity, normal is 7.35 - 7.45
• 1915: Lewisohn
o determined the minimum amount
of citrate needed for
anticoagulation and demonstrated its
nontoxicity in small amounts
• 1916: Rous and Turner
o introduced a citrate-dextrose
solution for the preservation of
blood
- Function of glucose in RBC metabolism
was not understood until the 1930s
• February 1941: Dr. Charles Drew
o was appointed director of the first
American Red Cross Blood Bank
at Presbyterian Hospital
• 1943: Loutit and Mollison of England
o introduced the formula for the
preservative acid-citrate-dextrose
(ACD) – extends the shelf life of
blood up to 21 days
• July 1947:
o Journal of Clinical Investigation
(blood preservation)
• 1957: Gibson
o introduced an improved preservative
solution called citrate-phosphate-
dextrose (CPD)
Anticoagulant-Preservative Solutions
Phosphate – maintains pH
Citrate – anticoagulation
• 1666: Richard Lower
o First successful animal to animal
transfusion using dogs
o 1667: Also performed animal
(sheep) to man (Arthur Coga)
transfusion
• 1667: Jean-Baptiste Denis
o first documented animal to man
transfusion
o successfully transfused sheep blood
into a 15y/o boy with a long-standing
fever
 o HLA System (Human Leukocyte
Antigen)
- Glycoplipid
o ABH, Lewis, Ii, P
-

BASIC BLOOD BANKING PRINCIPLES B. ANTIBODY

a) Antigen - Glycoprotein (immunoglobulin) that
b) Antibody recognizes a particular epitope on an
c) Antigen-antibody reactions antigen and facilitates clearance of
that antigen
A. ANTIGEN - Gamma globulin
- Foreign molecules that bind
specifically to an antibody or to a T-
cell receptor
o Immunogen – antigen in its role
of eliciting an immune response
2 types of antigens
1. Self Ag – tolerated, no immune
response
2. Foreign Ag – must be eliminated
Characteristics of Antigens: Properties
that Influence Immune Response
▪ Size – the bigger, the more IgE
immunogenic IgA
▪ Complexity – the more complex, the IgD
more immunogenic IgM
▪ Conformation IgG
▪ Charge - IgM, IgG most significant
▪ Accessibility
▪ Solubility
▪ Digestibility
▪ Chemical composition
- Proteins blood group
o Rh, MN
- Glycoproteins
 IgG
- Reacts at body temperature – 37degC
warm ab/agglutinin
- IgM – star shaped; crab shaped (unbound)
- Capable of destroying transfused antigen-
positive RBCs
- Predominant Ab produced in the secondary
response
- monomer
- Subclasses: IgG1, IgG2, IgG3, IgG4
- Rh, Duffy, Kidd, Kell
- Immune antibody – produced after an
exposure (blood transfusion, pregnancy)

pentamer monomer
Complement – naturally occurring plasma protein

Phases in Cross Matching

• Immediate Spin (IS) – detect IgM
• Thermo Phase
• Anti Human Globulin Spin Longer shorter

IgM – first antibody produced in immune response IgA

- Mucous membrane
IgM - Exists as monomer, dimer or trimer joined by
- Most commonly encountered naturally J chain
occurring Ab (ABO system) - 30% of anti-A and anti-B are IgA Abs
- Reacts at ambient temperature (22-24degC) - May cause severe anaphylaxis if transfused
- Cold agglutinin in IgA-deficient patients
- Produced in response to commonly occurring - Can increase the effect of IgG-induced RBC
antigens hemolysis
o Intestinal flora and pollen grains
- ABO, Lewis, Ii, P, MNS IgE
- Can interfere with
detecting IgG by - May cause urticaria if transfused in patients
masking their with severe allergic reactions
reactivity o Due to release of histamines
- Can exist in
monomeric or IgD
pentameric form - Least significant in blood banking
with J chain -> - Not able to cross placenta and activate
secretions complement
- Play a significant role in B cell activation and
expression

➢ Polyclonal
- Antibodies derived from more than one
antibody-producing parent cell
- Produced in response to a single antigen with
more than one epitope
➢ Monoclonal
- Antibodies derived from a single ancestral
antibody-producing parent cell
- Preferred in testing: highly specific, well
characterized, and uniformly reactive
- Anti-serum = anti A, anti B
- Used in test kits

❖ Naturally Occurring
 - Found in individuals without previous 4. Tolerance
exposure to RBC Ags from transfusion,
injection or pregnancy
- Mostly IgM cold agglutinins 1. Intermolecular Binding Forces
o Reacts in saline at ambient temp or
at 4oC
o May cause hemolysis at 37oC
- Commonly encountered: ABH, Hh, Ii, Lewis,
MN, and P blood group systems
❖ Immune
- Found in individuals with previous
exposure to transfusion, injection and 2. Antibody Properties
pregnancy Affinity – strength of the binding between a single
- Mostly IgG antibody and an epitope of an antigen
o Reacts best at 37oC Avidity – overall strehth of reaction between several
o Requires AHG (Coomb’s) sera for epitopes and antibodies; depends on the affinity of the
detection antibody, valency, and noncovalent attractive forces
- Commonly encountered: Rh, Duffy, Kidd, Kell, Valency – number of epitopes per molecule of antigen
ss blood group systems 2 valence IgG 10 valence IgM
• the specificity of an antiserum (or antibody) is
Alloantibodies one of its most important characteristics and
- Produced after exposure to non-self- is related to its relative avidity for antigen
antigens
Example: Rh neg exposed to Rh positive (D
antigen), you will produce anti-D
Autoantibodies
- Produced in response to self-antigens
- Transfused px with undetectable alloAb may
elicit stronger immune response against
previous Ags and cause severe transfusion
rxns Cross reaction – 2 Ag share the same glucolipid
- Positive auto control or direct antiglobulin test antigenic sites
(DAT)
Patient serum + Patient RBC = if reacts 3. Host Factors
positively, it will agglutinate = autoAbs

C. ANTIGEN-ANTIBODY REACTIONS
• When the antigen and antibody
combine, an antigen-antibody
complex or immune complex is
produced
Ag + Ab = Ag-Ab
o Immune complex: complex of
one or more antibody molecules
bound to an antigen
• Lock and key mechanism – extent of
the reciprocal relationship (fit) between
the antigen and its binding site on the 4. Tolerance
antibody - Defined as the lack of an immune
response or an active immunosuppressive
response
o Chimera – exposure to an antigen
during fetal life
o Preventing d-negative mothers from
developing anti-D antibodies after
delivering Rh-positive infants

Factors influencing antigen-antibody reactions:

1. Intermolecular binding forces
2. Antibody properties
3. Host factors
Antigen-Antibody Reactions in Vivo
• Transfusion, pregnancy, and the immune
response
o Patient’s immune system may
become activated or “sensitized”
with the resultant production of
circulating antibodies due to
exposure to foreign antigens
• Complement Proteins
o Classical pathway – antigen-
antibody reaction
o Alternative pathway – membrane
property of a microorganism
Clearance of Antigen-antibody Complexes
- Removed from the body’s circulation through
the mononuclear phagocyte system
Antigen-Antibody Reactions in Vitro
• In vitro RBC Ag—Ab reactions are detected
by visible agglutination (hemagglutination)
or hemolysis
• Stages of Hemagglutination
o Sensitization
▪ Attachment of Ab to
corresponding Ag on RBC
membrane
o Lattice formation
▪ Combination of antibody
and a multivalent antigen to
form crosslink
• Factors that influence agglutination
reactions:
a. Centrifugation
b. Ag-Ab Ratio
c. pH
d. Temperature
e. Ig type
f. Enhancement media
a. Centrifugation
- Enhances agglutination reactions
- Decreases reaction time by increasing the
gravitational forces on the reactants to
bringing reactants closer together
b. Ag-Ab Ratio
- Postzone reaction: Ag excess
- Prozone reaction: Ab excess
c. pH
- ideal pH for Ag-Ab reactions: 6.5-7.5
- anti-M and anti-Pr(Sp1)
o reacts at lower pH
d. Temperature
- Cold reacting: IgM type
o ABO Abs
- Warm reacting: IgG type
o Rh Abs
e. Ig Type
- IgM can readily agglutinate RBCs in saline
than IgG
- IgM has more Ag-combining sites than IgG
f. Enhancement media
Zeta Potential
- Difference in negative charge between the
inner and outer surfaces of the cloud
- RBCs are surrounded by negative ions when
they are suspended in isotonic saline
- high ZP = high distance = more difficult for
RBCs to agglutinate
Protein Media
- decreases ZP by increasing dielectric
constant or adding more cations
- albumin, PEG, polybrene,
polyvinylpyrrolidone (PVP), protamine
Low ionic strength solution (LISS)
- low salt media: 0.2% NaCl
- high rate of Ab uptake = low incubation time
o 30-60mins → 5-15 mins
Proteolytic enzymes
- Used in blood banking:
o Ficin: isolated from fig plants
o Papain: papaya
o Trypsin: pig stomach
o Bromelin: pineapple
- Cleaves sialic acid from RBc membrane →
low negative charged and low ZP
- Enhanced: Rh, Kidd, P1, Lewis, and I Ags
- Depressed: MNSs, Duffy Ags
Anti-human globulin (AHG) reagents
- Determine if RBCs are coated with Ab and/or
complement
- Small IgG antibodies can sensitize but rarely
cause agglutination.
o Cannot overcome zeta potential
UNIT 2: ABO BLOOD GROUP SYSTEMS

A. INTRODUCTION

ABO Blood Group

- Most important system in transfusion and
transplantation therapy
- The only blood group system in which
individuals have antibodies in their serum to
antigens that are absent from their RBCs
RBC surface → antigen
serum → antibodies
- 1st human blood group system
- Discovered by Karl Landsteiner in 1901
- ABO testing
o Most frequently performed test in the
blood bank
o Forward and reverse grouping
o Slide or tube
• Forward/Cell/Direct Typing
- unknown Ag, Rgt is known anti-
sera
• Reverse/Serum/Backward Typing
- Known RBC suspensions, Characteristic of routine reagents Used for ABO
unknown abs Testing

Inverse reciprocal – if antigen is present, ab is absent

Anti A- blue (Thymol blue)

Anti B – yellow (acriflavine)
cold agglutinin
4-5% RBC suspension (tomato red)
 ABO Antibody Production
Gel technology Initiated at birth
- Need centrifuge with same size - Low titer detection until infants are 3 to 6
months old
- Most antibodies in cord blood serum are of
maternal origin
- Perform only forward grouping
Peaks between 5 and 10 years of age and declines
later in life
- Low levels of antibodies in elderly people
- Antibodies may be detectable in reverse
grouping

H antigen – needed to form A and B antigen

C. INHERITANCE OF THE ABO BLOOD GROUPS

ABO Inheritance
• 1st described in 1924
• “An individual inherits one ABO gene from
each parent and these two genes
determine which ABO antigens are
present on the RBC membrane”
• Follows mendelian genetic law

A1> A2
AB>O
A=B

Frequency of ABO blood groups

O – most frequent
AB – least frequent

B. ABO ANTIBODIES

Characteristics of ABO Antibodies

• Naturally occurring in plasma
o Produced without any exposure to
RBCs
o Immune ab -> abs are produced bc
of exposure to a foreign ag
• Predominantly IgM – cold agglutinin,
pentamer
• Activate complement
• React at room temperature or colder
• Produce strong agglutination reactions Formation of ABH Antigens
 • Interaction of ABO, Hh, and Se
o Produce specific
glycosyltransferases that add
sugars to the precursor substance
(enzyme)
• Paragloboside or glycan
o Basic precursor material from which
A, B, and H antigens originate
• Inherited gene elicits specific enzyme
transferases which attach sugars to the
paragloboside

ABH Antigens
• Develop early in fetal life
• Expression of A and B antigens on the RBCs
o Fully developed by 2 to 4 years of
age
o Remains constant throughout life
• Phenotypic expression may vary with
o Race H Gene
o Genetic interaction • Present in more than 99.99% of the random
o Disease states population
o “h” is quite rare
On the 37th day of fetal life, attachment of o “hh” is extremely rare
immunodominant sugars occur on the RBC membrane ▪ Does not elicit production of
and it is dependent on ABH genes inherited a-2-L-fucosyltransferase
• Bombay phenotype
o Inherit ABO genes
o Lacks normal expression of ABH
antigens
o Inheritance of the “hh” genotype

H Antigen
• Precursor structure on which A and B
antigens are made
• Results from the inheritance of the H gene
• Inheritance of H and Se genes
o Influences A and B antigen
expression
o Located on chromosome 19
o H gene: inherited to form ABO
antigens on the RBCs
o Se gene: inherited to form ABO Blood Group A Formation
antigens in secretions • A gene
o Codes for α-3-N-
Interaction of Hh and ABO Genes acetylgalactosaminyltransferase
• Immunodominant sugars o Elicit higher concentrations of
o Sugars occupying the terminal transferase than the B gene
positions of the precursor chain • 810,000 to 1,170,000 antigen sites exist on
o Conferring blood group specificity A1 adult RBC
• L-fucose
o Sugar responsible for H specificity Blood Group B Formation
o Must be formed for the other sugars • B gene
to be attached in response to an o Codes for a-3-D-
inherited A and/or B gene galactosyltransferase
 • 610,000 to 830,000 antigen sites exist on a B
adult RBC
ABO Blood Group
• A and B genes are inherited
• 2 immunodominant sugars
• B enzyme seems to compete more efficiently
for the H substance than the A enzyme
o A antigen on an AB cell: 600,000
sites
o B antigen on an AB cell: 720,000
sites
Formation of ABH Soluble antigens
• ABH antigens
o Integral part of the membranes of
RBCs, endothelial cells, platelets,
lymphocytes, and epithelial cells
• ABH-soluble antigens
o Found in all body secretions
o Dependent on inherited ABO genes
and secretor genes (Sese) (SeSe,
Sese)
if sese = non secretor
Zz system
- Regulates production of H antigens on
erythrocytes
Sese system
- Regulates the formation of H antigen and
subsequently, of A and B antigens in
secretory cells
Zz Sese
RBC Secretions
Se Gene
• Codes for the production of α-2-L-
fucosyltransferase
• If present, the H substance can be further
modified
o Expressing A and B substance in
secretions such as saliva
• Genotype “sese” are non-secretors
• Secretor refers only to secretion of ABH
soluble antigens in body fluids
• Example: group A who is a secretor (SeSe or
Sese)
o Will secrete glycoproteins carrying A
and H antigens
o Se gene does not affect the ABH
antigen
• Presence of this gene determined whether
abh-soluble substances will be secreted
Saliva Test
Hemagglutination-inhibition
No agg → + result
Agg → - result
Procedure:
- Collect saliva
- Heat saliva to remove enzymes
- Centrifuge
- 2 layers: precipitated and supernatant at top
- Get supernatant and dilute 1:2
Anti-H is from ulex europeaus lectin
ABH Substances in Saliva
• Demonstration of ABH substances in saliva is
evidenced of the inheritance of an A gene, B
gene, H gene, and Se gene
Comparison of ABH antigens of RBC and ABH
Soluble Substances
 A1 and A2 Subgroups
• 80% of all group A (or AB) individuals are A1
(or A1B)
• 20% are A2 (or A2B) or weaker subgroups
• 1% to 8% of A2 individuals produce anti A1
in their serum
• 22% to 35% of A2B individuals produce anti-
A1

• Both are formed due to attachment of an

immunodominant sugar to an oligosaccharide
chain
o Type 1 and 3 chains: associated
with body secretions
o Type 2 and 4 chains: associated
with RBC membrane
o Type 1 and 2 chains: more
abundant; differ only in the linkage
position of galactose to N-
acetylglucosamine
A1 and A2 Genes
• A1 gene creates between 810,000 and
1,170,000 antigen sites on the adult A1 RBC
• A2 gene results in production of only 240,000
to 290,000 antigen sites on the adult A2 RBC
• Immunodominant sugar: N-acetyl-D-
galactosamine

D. ABO SUBGROUPS
A1 Antibody
A Subgroups • Naturally occurring IgM cold-reacting antibody
• Described by Von Dungern in 1911 • Unlikely to cause transfusion reaction
• Two different A antigen based on reactions o Because it usually reacts only at
between group A RBCs and anti-A and anti- temperatures <37degC
A1 • Clinically significant if reactive at 37degC
• More common than B subgroups
Relationship with H antigen

H antigen
- Found in greatest concentration on the RBCs
of group O individuals
- May not be detectable in group A1 individuals
o In the presence of the A1 gene,
almost all of the H antigen is
converted to A1 antigen
 o Placing the N-acetyl-D-
galactosamine sugar on the H
substance
o Due to the presence of many A1
antigens, H antigen on A1 and A1B
RBCs may be hidden
- In the presence of an A2 gene, only some of
the H antigen is converted to A antigens
o The remaining H antigen is
detectable on the cell

Reactivity of Anti-H Antisera (or Lectin) with ABO

E. THE BOMBAY PHENOTYPES
blood Groups
Bombay Phenotype (Oh)
- 1st reported by Bhendee in 1952 in
Bombay, India
- Phenotype results from inheritance of a
double does of h gene
Lectins - Inherited as an autosomal recessive trait
- Seed extracts that agglutinate human cells o Mutation in the FUT1 (H gene) and
with some degree of specificity FUT2 (Se gene) producing a
silenced gene
Para-Bombay Phenotypes
- Rare phenotypes
- RBCs either completely lack H antigens or
have small amounts of H antigen present
- Express weak forms of A and B antigens,
which are primarily detected by adsorption
and elution studies
o Mutated FUT1 (H gene) with or
without an active FUT2 (Se gene) or
o Silenced FUT1 gene with an active
FUT2 gene
- Low level of H
F. ABO DISCREPANCIES
• Occur when unexpected reactions are
obtained in the forward and/or reverse
grouping
• Can be due to problems with the patient’s
serum, patient’s RBCs, or both
• Unexpected reactions may be due to extra
positive reaction or a weak or missing
reaction
• Must be resolved prior to reporting a patient
or donor ABO group
Technical Errors Causing ABO Discrepancies
Resolution
• Use saline suspension
• Obtain adequate information regarding the
patient’s age, diagnosis, transfusion history,
medication, and history of pregnancy
• New sample must be drawn and repeat the
test
• May administer group O, Rh-compatible
RBCs
Take note: Generally, in ABO discrepancies, RBCs
and serum grouping reactions are very strong and
weaker reactions typically represent the discrepancy
Group I Discrepancies
- Most common
- Unexpected reactions in reverse grouping
- Weakly reacting or missing antibodies
Can be seen in:
✓ Newborns
✓ Elderly patients
✓ Patients with leukemia or lymphoma
demonstrating hypogammaglobulinemia
✓ Patients using immunosuppressive drugs that
yield hypogammaglobulinemia
✓ Patients with congenital or acquired
agammaglobulinemia or immunodeficiency
diseases
✓ Patients with bone marrow or HPC
transplants
Resolution: Group I
• Obtain patient clinical history
o If elderly individual or has
hypogammaglobulinemia
▪ Incubate serum with
reagent A1 and B cells at
RT for 15-30 minutes
▪ If no reaction after
centrifugation, serum-cell
mictures can be incubated
at 4degC for 15-30 minutes
 • Auto control and O cell control must always
be tested with reverse typing
• Investigate patient’s transfusion records and
clinical history to verify mixed-field
agglutination
o Mixed-field agglutination: small to
large agglutinates with agglutinated
cells
o May appear as “halo” or “puff of
smoke”
o Causes include receiving non-ABO-
type specific RBCs, ABO subgroups
(A3), bone marrow or hematopoietic
stem cell transplant
Group II Discrepancies
• Probably the least frequently encountered
o Unexpected reactions in the
forward grouping
o Weakly reacting or missing antigens
Causes:
✓ Subgroups of A or B may be present
✓ Leukemias may yield weakened A or B
antigens
✓ Acquired B phenomenon
o Bacterial enzymes modify N-acetyl-
D-galactosamine into D-
galactosamine
Resolution: Group II
• Incubation at RT for 30 minutes
o If reaction is still negative, incubate
at 4oC for 15-30 minutes
• Include group O and autologous cells as
controls
• RBCs can be pretreated with enzymes and
retested with antisera
• Test the patient’s serum or plasma against
autologous RBCs
• Secretor studies can be performed to
characterize the acquired phenomenon
Group III Discrepancies
• Discrepancies between forward and
reverse groupings
o Caused by protein or plasma
abnormalities
o Result in rouleaux formation or
pseudo agglutination
▪ Rouleaux: stacking of
RBCs in a coin-like fashion
Causes:
✓ Elevated levels of globulin from certain
disease states
o Multiple myeloma, waldenstrom’s
macroglobulinemia, other plasma
cell dyscrasias, and certain
moderately advanced cases of
Hodgkin’s lymphomas
✓ Elevated levels of fibrinogen
✓ Plasma expanders
✓ Wharton’s jelly in cord blood samples
Resolution: Group III
• Saline replacement technique
o Remove the serum and replace by
an equal volume of saline
o True agglutination: RBC clumping
remains after addition f saline
• Cord blood samples
o Thorough washing (6-8x) with saline
Group IV Discrepancies
• Discrepancies between forward and
reverse groupings
o Due to miscellaneous problems
Causes:
✓ Cold reactive autoantibodies
 ✓ Circulating RBCs of more than one ABO
group due to RBC transfusion or marrow/stem
cell transplant
✓ Unexpected ABO isoagglutinins
✓ Unexpected non-ABO alloantibodies

Resolution: Group IV
• Incubation of red blood cells at 37oC, wash
with saline at 37oC (3x) then retype
o If not resolved, treat RBCs with 0.01
M dithiothreitol (DTT)
• Reagent RBCs and serum can be warmed to
37oC for 10-15 minutes, mixed, tested, and
read at 37oC.
• Cold auto absorption could be performed
• Perform antibody identification panel
UNIT 3: RH BLOOD GROUP SYSTEMS
Rh Blood Group System
- Consists of over 50 related antigens
- The genes that control the system are located
in chromosome 1
- Polymorphic
- Only the most clinically significant will be
emphasized:
o D, C, E, c, and e
Importance of the Rh system
- After the A and B antigens, the D antigen is
the most important red cell antigen in blood
banking
- The D antibody can cause transfusion
reactions and hemolytic disease of the
newborn (HDN)/Erythroblastosis fetalis
History
- 1939 Levine and Stetson defined D antigen
(Rh factor)
o Levine and Stetson described a
hemolytic transfusion reaction in an
obstetric patient following delivery of
stillborn infant. The women required
transfusion. Her husband, who had
the same ABO type, was selected as
her donor, after transfusion the
recipient, demonstrated the classic
symptoms of acute hemolytic
transfusion reaction.
- Subsequently an antibody was isolated from
the mother’ s serum that react both at 37 °C
and 20 °C with the father’ s red cells. It was
postulated that the fetus and the father
possessed a common factor that the mother
lacked
- While the mother carry the fetus, the mother
was exposed to this factor and subsequently
built up an antibody that reacted against the
transfused red cellsfrom the father and
resulted in hemolytic transfusion reaction.
- 1940 Landsteiner and Wiener discovered
anti-Rh (named after Rhesus monkey)
o Agglutinated 85% human RBCs
o 15% RBCs did not agglutinate
- Landsteiner and Wiener reported on an
antibody by guinea pigs and rabbits when
they were transfused with rhesus monkey red
cells.
- This antibody which agglutinated 85% of
human red cells was named Rh.
- The name Rh was retained for the human
produced antibody.
- Anti-rhesus formed by the animals was
renamed anti-LW (Landsteiner and Wiener).
- Discovery of the Rh system:
o Levine and Stetson linked the Rh
factor to HDN
o Wiener linked Rh factor to
transfusion reactions
Rh Antigen Frequency
- D antigen -85%
- C antigen – 70%
- c antigen – 80%
- E antigen – 30%
- e antigen – 98%
Rh Genetics
- 2 closely linked genes control the expression
of ALL Rh antigens (codominant alleles)
o RHD gene - determines the
expression of the D antigen
RHCE gene - determines the expression of the C, c, E,
and e antigens
Rh Nomenclature
- There are several different systems of
nomenclature that theorize the inheritance of
the Rh system
o Fisher-Race
o Wiener
o Rosenfield
The terminology used to describe the Rh system is
derived from 4 sets of investigators.
Two of the terminologies are based on the postulated
genetic mechanisms of the Rh system.
3rd terminology describes only the presence or
absence of a given antigen.
4th is result of the effort of the International Society of
Blood Transfusion (ISBT) Working Party on
Terminology for Red cell Surface antigens.
Fisher-Race Nomenclature
- CDE terminology
- Most commonly used
- Developed by Ronald Fisher and Robert
Race of England
- They theorized that the Rh antigens are
controlled by a complex of 3 sets of genes
with closely linked loci (i.e. Dce gene complex
codes for D, c, e antigens)
Fisher-Race
 - There are 8 gene complexes at the Rh locus

Wiener
- Wiener further theorized that 8 major genes
led to different combinations of antigens (D,
C, E, c, e):
o Rh0, Rh1, Rh2, Rhz
- Fisher-Race uses DCE as the order o rh, rh’, rh”, rhy
- Others alphabetize the genes as CDE
Wiener Genes and Antigens
Fisher-Race Example
- DCe/DCe individual is homozygous for D, C,
and e genes
- DCe/dcE individual is heterozygous for D, C,
e, d, c, and E genes

Wiener Nomenclature
- Rh-Hr terminology
- Rarely used, but good for describing
phenotype
- A single gene at the Rh locus leads to the
expression of the Rh antigens
- Each parent contributes 1 Rh gene Converting Wiener into Fisher-Race or vice versa
- 8 alleles exist at each gene locus
- Each gene controls production of an
agglutinogen composed of three factors
(antigens)

Wiener Theory

Differentiating superscript from subscript

- Superscripts (Rh1) refer to genes
- Subscripts (Rh1) refer to the agglutinogen
(complex of antigens)
- For example, the Rh1 gene codes for the Rh1
agglutinogen made of D, C, e
o Usually, this can be written in
shorthand, leaving out the “h”
o DCe is written as R1

Who was right?

 - Both Fisher-Race and Weiner were on the
right track
- Molecular genetics has shown that both
theories were partially right
o Weiner theorized that the genes
have multiple specificities
o Fisher-Race theorized that there
were more than one gene (3 to be
exact!)
R1r or Dce/dce – most common in whites approx. 35%
R0r or Dce/dce – most common in blacks approx. 42%
Rosenfield Nomenclature
- Antigens are designated by number
o Rh1:D
o Rh2:C
o Rh3:E
o Rh4:c
o Rh5:e
o Example
▪ D+, C+, E-, c+, e+ is written
as Rh:1,2,-3,4,5
ISBT
- International Society of Blood Transfusion
(ISBT)
- Attempts to standardize nomenclature
- Six digit numbers are assigned to each blood
group specificity
- 004 refers to the Rh system
- The remainder is the Rosenfield system
number (i.e. C antigen is RH2)
- Example: ISBT for the C antigen is 004002
LW – is the antigen closely associated phenotypically
with Rh
- It is formerly known as Rh25
Rh locus – located on chromosome 1 (along with the
genes for elliptocytosis)
Genotype vs. Phenotype
- The phenotype is the result of the reaction
between the red cells and antisera
- The genotype is the genetic makeup and can
be predictd using the phenotype and by
considering race of an individual
- Only family studies can determine the true
genotype
Phenotype
- Anti-D, anti-C, anti-E, anti-c, and anti-e is
tested with patient RBCs
- If a specimen gives the following reaction: D+,
C+, E-, c+, e+
- The phenotype would be DCce
- The most probable genotype would be
o DCe/dce
o DCe/Dce
Probable genotype
- If the RBCs express both C and c or E and e,
the corresponding genes are present in the
heterozygous state
- If they express only C or c, or only E or e, the
person is assumed to be homozygous for that
gene
Rh antigens and antibodies
Rh Antibodies
- Are IgG, react optimally at 37 oC or following
the addition of antiglobulin reagent (IAT)
- Produced after exposure of the individual’ s
immune system to foreign red cells, either
through transfusion or pregnancy.
- Rh Abs often persists in the circulation for
years
- Rh Abs are considered clinically significant,
therefore, Ag negative blood must be
provided to any patient with a history of Rh Ab
sensitization.
- Rh Abs do not bind complement. For
complement to be fixed, two IgG molecules
must attach in close a proximity on the red
cell surface.
 - Red cell destruction due to Rh Abs is
primarily extravascular. This type of
hemolysis classically characterizes a delayed
hemolytic transfusion reaction.

Rh Antigens
- Rh antigens are highly immunogenic, the D
antigen is most potent
- D > c > E > C > e
Highly -----------→ rarely
Immunogenic
- Exposure to less than 1 ml of Rh positive red Variation of the Rho (D) Antigen
cells can stimulate Ab production in an Rh - Weak D (Du Ag)
negative person. o When Rh-positive red cell samples
are typed for the D Ag. It is expected
Varieties of Rh antibodies that they will react strongly with anti-
1. First order Rh abs : react strongly with D reagent.
specific Rho ags in saline medium & react o However, with certain red cells the
less strongly in a protein medium testing must be carried through the
2. Second order Rh abs: react visibly with AHG phase to demonstrate the
specific ag in a protein medium. In saline, presence of the D Ag.
weakly combine with the ag but do not o Red cells carrying the weaker D Ag
produce a visible reaction have been referred to as having the
3. Third order Rh abs: react visibly with specific Du type.
ags in the antiglobulin medium only
DETERMINATION OF D STATUS
Determination of D Status
- Is essential when testing donor blood sample.
- Blood considered Rh positive if either the D or
Du test is positive
- If any donor blood sample that types Rho(D)
negative by either slide or rapid method must
be tested further by indirect anti-globulin test
Weak D (Du Ag)
(IAT).
- Three different mechanisms have been
- If both test results are negative, the donor
described that can explain the weakened
sample is considered Rh negative.
expression of the D Ag. ¢
- Genetic weak D
Determination of Du Status
o Inheritance of D genes that code for
There are instances when an accurate Rh type
can not be determined through routine testing a weakened expression of the D Ag.
- 1 – If the newborn’s cell are located with o The D Ag expressed appear to be
maternal IgG anti-D in utero, very few D Ag complete but few in number.
sites will be available to react with reagent Inheritance of these gene can be
anti-D. tracked from one generation to the
- Elution of the sensitizing Ab 9removing the next and seen most frequently in
Ab) and identifying it as anti-D will verify that black.
the infant’s red cell are D positive Weak D – Genetic
- 2 – Warm autoimmune hemolytic anemia,
Abs are directed against the patient’s own red
cell and react as through they are Rh specific.

Rh Genetics
- 2 closely linked genes control the expression
of ALL Rh antigens (codominant alleles)
o RHD gene - determines the
expression of the D antigen
o RHCE gene - determines the
expression of the C, c, E, and e
antigens
Variation of the Rho (D) Antigen
- C Trans (a position effect or gene interaction
effect)
- The Rh Ag on the red cell is normal, but the
steric arrangement of the C Ag in relationship
to the D Ag appears to interfere with
expression of the D Ag.
- This interference with D expression does not
occur when the C gene is inherited in the
cis (The location of two or more genes on the
same chromosome of a homologus pair)
.Family study can be distinguish which type of
weakened D Ag is being demonstrated.
Test for Weak D Antigen (Du)
Procedure
Position Effect 1. Prepare 2-5% red cell suspensions to be
tested
2. Label another set of tubes as U and NC
3. Follow the table below.
CONTENT Unknown Negative Control
Anti-D 1 drop -
22% Bovine - 1 drop
Serum Albumin
2-5% red cell 1 drop 1 drop
suspension

4. Mix gently and cover all tubes with Nescofilm.

Variation of the Rho (D) Antigen
- Partial D
o One or more parts of the D Ag are
missing or altered Wiener and Unger
postulated that the D antigen is
made of antigenic subparts,
genetically determined, that could be
absent in rare instances.
o If an individual lacked one (or more)
pieces, or epitopes, of the total D
antigen, alloantibody can be made to
the missing epitope(s) if exposed to
RBCs that possess the complete D
antigen. This theory has become
well accepted.
D Mosaic/Partial D
- If the patient is transfused with D positive red
cells, they may develop an anti-D
alloantibody* to the part of the antigen
(epitope) that is missing
Incubate both tubes for 15 minutes at 37°C
water bath. Unknown tube with agglutination
is regarded as Rh positive. Unknown Tube
without agglutination goes to the next
procedure.
5. Wash the cells 3 times with normal saline
solution.
6. Decant the saline completely after the final
washing.
7. Add 2 drops of antihuman globulin reagent to
all tubes and mix gently. Cover with
Nescofilm.
8. Centrifuge for 15 seconds at 3400 rpm.
9. Gently dislodge each cell button and examine
for agglutination.
10. Interpret and record results.
11. For each negative tube, add 1 drop of well
mixed check cells.
Note: Check cells or Coomb’s cells are RBCs coated
with IgG. A positive resyult is expected after the
addition of check cells. His implies that the test has
been properly performed. Negative result with these
cells indicates an improperly performed test and the
test should be repeated.

Guide in the Interpretation of Tezt for Weak D

Antigen
Anti-D Manner of ADDITIONAL
- Alloantibody – antibody produced with (slide or tube Reporting COMMENT
specificity other than self method)
+ Rh Positive Positive
reaction
indicates
 presence of D-
antigen
0 Rh Negative Negative
(initial) rection should
be further
tested for the
presence of
weak D with the
use of AHG
[ + ] with agglutination [ 0 ] no agglutination
AHG Test for Weak D: (Indirect Antihuman globulin
Test)

Du Reaction Manner of ADDITIONAL Unusual Phenotypes/Rare Alleles

Reporting COMMENT - Cw
0 Du negative Rh(-) Du (-): - f (ce)
Negative - rhi (Ce)
reaction - G
confirms that
- Rh 13, 14, 15, and Rh 16
the person is
Rh negative. - Rh 17 (Hr0)
(patient or - Rh 23, Rh 30, Rh 40
donor). - Rh 33 (Har)
+ Du positive Rh(-) Du (+) - Rh 32
should be given - Rh 43 (Crawford)
proper - E variants
classification:
a. If Patient: Rh - V and VS
Negative G Antigens - are produced by the same Rh gene
b. If donor: Rh complexes that produce C and D antigens. Most C-
Positive positive and D-positive RBCs are also G-positive
[ + ] with agglutination [ 0 ] no agglutination f(ce) – a compound antigen of c and e
Other Phenotypes rhi(Ce) – a compound antigen of C and e
Del Rh Deficiency Syndrome: Rhnull and Rhmod
- Del is a phenotype occurring in individuals - Rhnull: individuals who lack Rh ags on the
whose red blood cells possess an extremely RBCs
low number of D antigen sites that most 2 genetic mechanisms
reagent anti-D are unable to detect. o Mutations in the RHAG gene
- Adsorbing and eluting anti-D from the (regulator type)
individual’s red cells is often the only way to o Amorphic type
detect the D antigen. This procedure requires - Rhmod: partial suppression of RH gene
incubating anti-D with the RBCs in question at expression caused by mutations in the RHAG
37°C followed by eluting the anti-D off the gene
adsorbed red cells. The 37°C incubation Rhnull SYNDROME
provides time for the anti-D to bind to the few It expresses no Rh antigen on red cell and the
D antigen sites present on these red cells phenotype is expressed as ---/---. The ff. are the
symptoms:
D epitopes on RhCE protein - Stomatocytosis
- Phenotypes: DHAR and ceCF (Crawford) - Reticulocytosis
- R0 Har / DHAR : results from a hybrid gene - Increased HbF, bilirubin
RHCE-RHD-RHCE where only a small portion - Decreased haptoglobin, OFT –
of RHD is inserted into the RHCE gene ¢ - slight ↓ Hb, Hct
ceCF(Crawford) : found in individuals of - Compensated hemolytic anemia
African descent; results from a specific Anti-Rh:29
amino acid change in the Rhce gene, - the antibody of broadest specificity
resulting in an RhD epitope on the Rhce - described as anti-total Rh
protein - can be produced by Rh null individuals
Deletions
- No Cc and/or Ee reactivity
- Have unusually strong D expression (exalted
D)
- Indicated by dash (-)
 - A variation has been recognized within the
deletion D–, called D••
- Some deletion phenotypes are missing E/e
only and are indicated as Dc- or DC- . To
date, a deletion of only C/c has not been
reported.
CLINICAL CONSIDERATIONS
- When Rh antibodies are identified, it is
important to understand clinical implications 2. Rouleaux
when Rh incompatibility exists
- TRANSFUSION REACTIONS
- HEMOLYTIC DISEASE OF THE FETUS AND
NEWBORN
Rh Typing Reagents
- Reagents used to type for D and other Rh
antigens
- High-protein based, low-proteinbased, saline-
based, chemically modified, monoclonal or
blends of monoclonals 3. Cold Autoagglutinins - px sample should be
Rh Reagents warmed to 37ºC and immediately retested.
- Coomb’ s cells (C.C)
o Control cellular used when AHG test False Negative Reactions may be caused by:
is negative. To confirm that washing 1. incorrect cell suspension. (cell suspension too
has been adequate and the anti- heavy or too light)
globulin reagent is reactive POST ZONE effect – antigen excess
- Control Cell PROZONE effect – antibody excess
2. improper procedure
o A group of O Rh positive donor’s
sample is mixed with 1:10 dilution of
PROZONE VS P OSTZONE
reagent anti-D and allowed to
incubate. After sensitization of RBC,
they are washed with saline and
suspended to a 5% RBC
suspension. These sensitized RBCs
then used to confirm the anti-IgG
activity of the AHG.
- Coomb’s cells (C.C)
o They can be used to ensure that
AHG test with neg results are not
false neg because of inactivation of
the AHG reagent. When AHG test is
Recipient Genotype: DCe/dce
neg , they should be free AHG Donor Genotype: DCE/dce
reagent in the test tube When the Question? What antibody will the recipient form after
CC are added, the free AHG in the transfusion of the blood from the above mentioned
test should cause agglutination of donor?
the sensitized RBCs. Answer:
o This positive reaction is a mixed field Refer to the data:
in nature because; half of RBCs in Antisera Reactions
the mixture lack IgG on their surface Anti-D +
and are free cells Anti-C -
o 2nd half of RBCs have IgG (CC) and Anti-E -
are agglutinated Anti-c +
Anti-e +
False Reactions with Rh Typing False Positive Which of the ff. is a possible genotype?
reactions may be caused by: a. R1r’
1. Positive DAT – most common cause of Rh- b. R1R1
c. R0r
typing (Du typing) discrepancies.