HISTORY

Karl Landsteiner
- Discovered the first human blood group system
- First to perform forward and reverse ABO typing
ABO Grouping
• Most frequently performed test in blood bank
• Forward & Reverse typing
- Inverse reciprocal relationship
- Must be performed on all donors and patients
• Type O and A: most common blood types
• Type AB: rarest blood type
• Subgroup A2: rarely found in Asians
ABO Antibodies
• “naturally occurring”- produced without any exposure to RBCs
• predominantly IgM (but small amount of IgG may be present)
- activate complement
- react at room temperature or colder
• Can cause rapid intravascular hemolysis
• ABO antibody production:
- initiated at birth (titers too low for detection until 3-6 months of
age)
- Peaks between 5-10 years of age and declines later in life
ABO Antibodies
Before 3-6 months Elderlies
•Anti-A and Anti-B
•Only forward typing may be
undetectable in
reverse typing
•Forward typing
 ABO Antibodies
BLOOD TYPE ANTIBODIES
Group A Anti-B
Group B Anti-A
Group O Anti-A; Anti-B; Anti-A,B
Group AB None
Inheritance of ABO Blood
Groups
•follows simple Mendelian genetics
•ABO is codominant in expression
•One position/locus on each chromosome 9 is
occupied by an A, B, or O gene
•Genotype - AA, BO and OO (etc.)
•Phenotype - Group A, B, AB and O
•O gene:
- considered an amorph
- does not elicit the production of a catalytically
active polypeptide transferase
- no detectable antigen is produced
•Group O – autosomal recessive
•Group AB – inheritance both A & B gene
FORMATION OF A, B & H RED CELL Antigens
• ABO, Hh, and Se – 3 genes that interact with each other to
produce specific enzyme glycosyltransferases that add sugars to
a basic precursor substance called paragloboside or glycan
• ABO genes are located on chromosome 9
• The FUT 1 (H) and FUT 2 (Se) genes- closely linked and located
on chromosome 19
• ABH antigens develop early in fetal life
• expression of A and B antigens fully develop by 2 to 4 years of
age and remains constant in life
 Precursor Substances
TYPE II (IV) TYPE I (III)
• RBCs • Secretions
• Beta 1  4 linkage • Beta 1  3 linkage
between terminal between terminal
galactose and N- galactose and N-
acetylglucosamine acetylglucosamine
Type II Precursor
 Interaction of Hh and ABO Genes
• H gene elicits the production of an enzyme called α-2-L-
fucosyltransferase that transfers the sugar L-fucose on the
terminal galactose of type 2 chains
• Immunodominant sugars- the sugars conferring blood group
specificity
• H antigen is the precursor structure on which A and B
antigens are made
Formation of
H Antigen
 FORMATION OF A, B, and H SOLUBLE
ANTIGENS
• ABH-soluble antigens can
also be found in all body
secretions through ABO
and Sese genes
• Se genes- codes for the
production of Se gene-
specified α-2-L-
fucosyltransferase
• Precursor substance= Type I
• SeSe or Sese = secretor
• sese = non-secretor
Type I Precursor
Comparison of A,
B, and H Antigens
on RBCs with A, B,
and H Soluble
Substances
FORWARD & REVERSE TYPING
FORWARD & REVERSE TYPING
FORWARD & REVERSE TYPING
 ABO SUBGROUPS
- Represents phenotypes that show weaker and variable
serologic reactivity with commonly used human polyclonal
anti-A and anti-B and anti-A,B reagents
 A Subgroups
• In 1911 von Dungern described two different A antigens
based on reactions between group A RBCs and anti-A and
anti-A1
 A Subgroups
•99% of Group A individuals are classified
into A1 and A2
- A1 (A1B) = 80%
- A2 (A2B) or weaker subgroups = 20%
A1 vs. A2
 Inheritance of A1 gene
A1
• very potent gene A1
creates between Production of high
810,000 and 1,170,000 concentrations of α-3-N-
antigen sites on the acetylgalactosaminyltransferase
adult A1 RBC, whereas
inheriting an A2 gene Converts almost all H precursor
results in production of structure to A1 antigens on the
only 240,000 to 290,000 RBCs
antigen sites on the
adult A2 RBC
 A2
• A2 allele characterized by:
- single base substitution at nucleotide 467
- single base deletion at nucleotide 1060 (1060delC) in exon
7
• alter the active site of the coding region and subsequently
change the specificity of the A glycosyltransferase
• 1-8% of A2 individuals produce anti-A1
• 22-35% of A2B individuals produce anti-A1
 Anti-A1
• a naturally occurring IgM cold-reacting antibody
• unlikely to cause a transfusion reaction since it reacts
only at temperatures well below 37°C
• clinically significant if it is reactive at 37°C
• can cause discrepancies between forward and reverse
ABO testing and incompatibilities in crossmatches
with A1 or A1B cells
• Anti-A1 Lectin (Dolichos biflorus)- differentiates A1 and A2
phenotypes
• Lectins- seed extracts that agglutinate human cells with
some degree of specificity
 H Antigen
A1 A2
• May not be detectable in • Only some of the H antigen is
Group A1 individuals converted to A antigens, and
• may not be available to react the remaining H antigen is
with anti-H antisera detectable on the cell
- Anti-H Lectin (Ulex europaeus) • increased reactivity with anti-H
• anti-H is occasionally found in Lectin (Ulex europaeus)
the serum of Group A1
individuals
 Anti-H
• naturally occurring IgM cold agglutinin
• reacts best below room temperature
• insignificant antibody because it has no
reactivity at 37°C
HOWEVER, high-titered anti-H:
• may react at RT and present a problem in
antibody screening procedures (since
reagent screening cells are group O)
• may also present a problem with
compatibility testing
 Advance Concepts
4 Different forms of H antigen
• Two unbranched straight chains (H1, H2)
- can be converted to Aa and Ab antigens, respectively, by
both A1 and A2 enzymes
• Two complex branched chains (H3, H4)
- can be converted to Ac and Ad antigens by A1 enzyme and
only very poorly by A2 enzyme
 Results
• more unconverted H antigens (specifically H3 and H4) are available
on group A2 RBCs, and only Aa and Ab determinants are formed from
H1 and H2 structures
• Some A2 individuals, Ac is extremely low and Ad is completely lacking
(individuals in whom one would likely find anti-A1 in the serum)
• 22-35% of A2B individuals would far more likely to lack Ac and Ad
components with subsequent production of anti-Ac and anti-Ad
(anti-A1)
Weak A Subgroups
 Characteristics of Weak A Subgroups
• Decreased number of A antigen sites per RBC
(resulting in weak or no agglutination with human
polyclonal anti-A)
• Varying degrees of agglutination by human anti-A,B
• Increased variability in the detectability of H antigen,
resulting in strong reactions with anti-H
• Presence or absence of anti-A1 in the serum
 Serologic Techniques
• Forward grouping of A and H antigens with anti-A, anti-A,B,
and anti-H
• Reverse grouping of ABO isoagglutinins and the presence of
anti-A1
• Adsorption-elution tests with anti-A
• Saliva studies to detect the presence of A and H substances

*Absence of a disease process should be confirmed before

subgroup investigation
 A3
• anti-A: mixed-field agglutination
• most anti-A,B: mixed-field agglutination
• No. of A antigens: 35,000/RBC
• Weak α-3-N-acetylgalactosaminyltransferase activity is
detectable in the serum, but with molecular
heterogeneity
• Anti-A1 may be present in serum of A3 individuals
• A substance is detected in the saliva of A3 secretors
 Ax
• Anti-A1: no agglutination
• Most Anti-A,B: agglutination
• No. of A antigens: 4000/RBC
• α-3-N-acetylgalactosaminyltransferase is not usually detectable in
the serum or in the RBC membrane
• Adsorption-Elution of Anti-A: without difficulty
• Anti-A1 is always present in serum of Ax individuals
• Routine secretor studies: only H substance in secretors
• Agglutination/inhibition studies: Presence of A substance
 Aend
• Some anti-A: very weak mixed-field agglutination
• Some anti-A,B: very weak mixed-field agglutination
• No. of A antigens: 3,500/RBC (no A antigens on
unagglutinated cells)
• No α-3-N-acetylgalactosaminyltransferase activity is
detectable in the serum and RBC membrane
• Anti-A1 may be present in some serum of Aend individuals
• Only H substance is detected in the saliva of Aend secretors
 Am
• anti-A: No or weak agglutination
• anti-A,B: No or weak agglutination
• No. of A antigens: 200-1,900/RBC
• A enzyme of either the A1 or A2 type is detectable in the
serum
• Adsorption-Elution of Anti-A: strongly positive
• Anti-A1 is absent in the serum of Am individuals
• A and H substance are detected in the saliva of Am secretors
 Ay
• anti-A: No agglutination
• anti-A,B: No agglutination
• No. of A antigens: 1,000/RBC
• Trace amounts of glycosyltransferase is detectable in the serum
• Adsorption-Elution of Anti-A: confirms presence of A antigens
• Anti-A1 is absent in the serum of Ay individuals
• A (below normal amount) and H substance are detected in the
saliva of Ay secretors
* A germline mutation of an A gene within a family
 Ael
• anti-A: No agglutination
• anti-A,B: No agglutination
• No. of A antigens: 700/RBC
• No activity of glycosyltransferase is detectable in the serum or
on RBC membranes
• Adsorption-Elution of Anti-A: confirms presence of A antigens
• Anti-A1 is present in the serum that reacts with A1 cells
• sometimes produce anti-A, which agglutinates A2 RBCs
• Only H substance is detected in the saliva of Ay secretors
Weak B Subgroups
B3, Bx, Bm, Bel
 Criteria for Weak B Phenotypes
Differentiation
• Strength and type of agglutination with anti-B, anti-
A,B, and anti-H
• Presence or absence of ABO isoagglutinins in the
serum
• Adsorption-elution studies with anti-B
• Presence of B substance in saliva
• Molecular testing
 B3
• anti-B: mixed-field agglutination
• anti-A,B: mixed-field agglutination
• activity of glycosyltransferase is detectable in serum, but not
in RBC membranes
• Anti-B is absent in the serum
• B substance is detected in normal amounts in the saliva of
B3 secretors
• the most frequent weak B phenotype
 Bx
• anti-B: weak agglutination
• anti-A,B: weak agglutination
• activity of glycosyltransferase is not detected in serum and RBC
membranes
• Adsorption-Elution of Anti-B: readily exhibited
• Weakly reactive Anti-B is present in the serum
• Routine secretor studies: Large amount of H substance in
secretors
• Agglutination/inhibition studies: Presence of some B substance
 Bm
• anti-B: no agglutination
• anti-A,B: no agglutination
• Low activity of glycosyltransferase is detected in serum and very
small amounts of activity in RBC membranes
• Adsorption-Elution of Anti-B: easily exhibited
• No Anti-B is present in the serum
• Routine secretor studies: Normal amount of B and H substance
in secretors
• Reported to be more frequent in Japan
Reduced activity of B enzyme in hematopoietic
tissue is clearly the defect causing the formation
of the Bm subgroup

Normal B plasma + Bm RBCs + UDP-galactose =

Bm RBCs  Normal group B RBCs
 Bel
• anti-B: no agglutination
• anti-A,B: no agglutination
• No activity of glycosyltransferase is detected in serum and RBC
membranes
• Adsorption-Elution of Anti-B: determines this extremely rare
phenotype
• inherited as a unique mutation in exon 7 of the B gene at the ABO
locus
• Weak Anti-B may be present in the serum
• Routine secretor studies: Only H substance in secretors
The BOMBAY Phenotypes (Oh)
OhA, OhB, OhAB
• was first reported by Bhende in 1952 in Bombay, India
• inheritance of a double dose of the h gene, producing
the very rare genotype hh, an autosomal recessive
trait
• mutation in the gene FUT1 (H gene), producing a
silenced gene
• also associated with a silenced FUT2 gene (Se gene)
• fail to react with anti-A, anti-B, and anti-H antisera, but
would phenotype as an O blood group
• do not react with the anti-H lectin (Ulex europaeus)
• Bombay serum contains anti-A, anti-B, anti-A,B, and
anti-H
• anti-H (IgM) can often be potent and reacts strongly at
37°C, capable of binding complement and causing RBC
lysis
• Only blood from another Bombay individual will be
compatible and can be transfused to a Bombay
recipient.
 Para-Bombay Phenotypes
• RBCs either completely lack H antigens or have small
amounts of H antigen present
• Two genetic basis:
1. mutated FUT1 (H gene) with or without an active
FUT2 gene (Se gene)
2. silenced FUT1 gene with an active FUT2 gene
 Mutated FUT1 gene para-Bombay
• α-2-Lfucosyltransferase enzyme activity is greatly
reduced
• very small amounts of H, A, and B antigens are
produced
- detected only using adsorption and elution techniques
 Mutated FUT1 gene para-Bombay
 homozygous inheritance of a mutant H (FUT1) gene:
• A or B, the respective enzymes can be detected, but
no H enzyme is detectable
• small amount of H substance on the RBC is completely
consumed by the A or B transferase present, resulting
in small quantities of A or B antigen being present on
the RBC with no detectable H antigen
• Anti-H is present in serum, but weaker than that of
Bombay phenotype
 Silenced FUT1 gene with the active FUT2
gene para-Bombay
• FUT2 (Se)-specified α-2-L-fucosyltransferase enzyme
produces H, A, and B type 1 antigens (from type 1
precursors) in secretions, including plasma
- These antigens are adsorbed onto RBC membrane
• RBCs have little or no A, B, and H antigens
• Adsorption and elution of anti-H: may reveal presence of
some H antigen on the RBC
 Silenced FUT1 gene with the active FUT2
gene para-Bombay
• not usually agglutinated by anti-A and anti-B, however,
some OhA RBCs can be agglutinated by anti-A,B and potent
examples of anti-A
• Anti-IH: weak H-like antibody reactive at low temperature &
is almost always present in the serum
• Normal levels of H substances are present in the saliva. A
and B substances are present in the secretions when A and B
genes are present
 ABH Antigens and Antibodies in Disease
• additional acquired pseudoantigens
• Progressively weaker reactions (depressed antigen strength)
- Often show a mixed-field agglutination (tiny agglutinates in
a sea of unagglutinated cells; “halo” or “puff of smoke” of
unagglutinated RBCs)
• The isoagglutinins (anti-A, anti-B, or anti-A,B) also may be
weak or absent demonstrating hypogammaglobulinemia (i.e
CLL, various lymphomas, etc.)
 ABH Antigens and Antibodies in Disease
• disorders of the lower intestinal tract- allows passage of the
bacterial polysaccharides from Escherichia coli serotype O86
(acquired B phenomenon in group A1)
• carcinoma of the stomach or pancreas- serum contains
excessive amounts of blood group–specific soluble
substances (BGSS) that may neutralize the antisera used
ABO Discrepancies
- unexpected reactions are obtained in the forward and/or
reverse grouping
- must be resolved prior to reporting a patient or donor ABO
group
 Technical Errors
• blood sample and test tube labeling errors
• failure to add reagents
• the addition of incorrect reagents or sample
*Serum and antiserum should always be added first, followed
by the patient or reagent RBCs
• Results must be recorded immediately after obtaining them
• Always examine reagent vials concurrently while performing
ABO testing
• Do quality control testing for possible contamination
 Resolution
• Repeat testing of the same sample using a saline
suspension of RBCs can usually resolve the ABO
discrepancy
• Review and correct all technical factors that may lead
to ABO discrepancy
• obtain adequate information regarding the patient’s
age, diagnosis, transfusion history, medications, and
history of pregnancy
 Resolution
*due to an error in specimen collection or
identification: a new sample must be drawn from the
patient and all RBC and serum testing repeated
• always remember that RBC and serum grouping
reactions are very strong (3+ to 4+) and the weaker
reactions typically represent the discrepancy
 Group I Discrepancies
• unexpected reactions in the reverse grouping due to
weakly reacting or missing antibodies
• more common than those in the other groups
• depressed antibody production or cannot produce the
ABO antibodies
 Group I Discrepancies
• Newborns
• Elderly
• leukemia or lymphoma
• immunosuppressive drugs
• congenital or acquired agammaglobulinemia or immunodeficiency
diseases
• bone marrow or hematopoietic progenitor stem cell (HPC) transplants
• ABO antibodies may have been diluted by plasma transfusion or
exchange transfusion
• ABO subgroups
 Resolution of Common Group I Discrepancies
• Obtaining patient clinical history may immediately resolve
this type of discrepancy
1. enhance the weak or missing reaction by incubating the
patient serum with reagent A1 and B cells at RT for
approximately 15-30 mins. then centrifuge
2. serum-cell mixtures can be incubated at 4°C for 15 to 30
minutes (auto control and O cell control must always be
tested concurrently with the reverse typing)
• Serum problems are more common
 Rare Group I Discrepancies: Chimerism
• presence of two cell populations in a single individual
• True chimerism- occurs only in twins and is rarely found
- In utero exchange of blood occurs due to vascular
anastomosis
- both blood groups are recognized as self
• Dispermy (two sperm fertilizing one egg) - patient or donor
has no history of a twin and indicates mosaicism
 Rare Group I Discrepancies: Chimerism

• Artificial chimeras- mixed-field cell population due to:

- Blood transfusions
- Transplanted bone marrow or hematopoietic
progenitor stem cells (HPC) of a different ABO type
- Exchange transfusions
- Fetal-maternal bleeding
 Group II Discrepancies
• unexpected reactions in the forward grouping due to
weakly reacting or missing antigens
• least frequently encountered
• Subgroups of A or B may be present
• Leukemias may yield weakened A or B antigens
• “acquired B” phenomenon will show weak reactions
with anti-B antisera and is most often associated with
diseases of the digestive tract
 Resolution of Common Group II Discrepancies
1. Weak reactions can be enhanced by incubating the
test mixture at room temperature for up to 30
minutes
2. If it’s still negative, incubate the test mixture at 4°C
for 15 to 30 minutes (include group O and autologous
cells as controls)
- RBCs can also be pretreated with enzymes and
retested with reagent antisera
 Acquired B Phenomenon
• bacterial enzymes modify the immunodominant sugar N-
acetylD-galactosamine into D-galactosamine, which is
sufficiently similar to the group B sugar (D-galactose) to cross-
react with anti-B antisera
- Anti-B antisera: demonstrates a weak reaction, often yielding a
mixed-field appearance
• pseudo-B antigen is formed at the expense of the A1 antigen
• Monoclonal anti-B clone (ES4)- strongly agglutinate cells with
the acquired B antigen due to lowered pH of reagent
• Treat RBCs with acetic anhydride to reacetylate the surface
molecules
 Rare Group II Discrepancies
• Blood group–specific soluble (BGSS) substances- excessive
amount of these substances in plasma will neutralize the
reagent anti-A or anti-B, and yields a false-negative or weak
reaction in the forward grouping
- occurs with certain diseases, such as carcinoma of the
stomach and pancreas
• Resolution: Washing the patient cells free of the BGSS
substances with saline
 Rare Group II Discrepancies
• Antibodies to low-prevalence antigens in reagent anti-A or
anti-B
- this additional antibody in the reagent antisera has reacted
with the corresponding low-prevalence antigen present on
the patient’s RBCs
- this phenomenon is only seen when human source
antiserum is used
• Resolution: repeat the forward type, using antisera with a
different lot number
 Group III Discrepancies
• between forward and reverse groupings- caused by protein
or plasma abnormalities and result in rouleaux formation or
pseudoagglutination
- multiple myeloma, Waldenström’s macroglobulinemia, other
plasma cell dyscrasias, and certain moderately advanced
cases of Hodgkin’s lymphomas
- Elevated levels of fibrinogen
- Plasma expanders, such as dextran and polyvinylpyrrolidone
- Wharton’s jelly in cord blood samples
 Resolution of Common Group III
Discrepancies
 Rouleaux- is a stacking of erythrocytes giving the
appearance of agglutination
• Forward grouping- usually accomplished by washing the
patient’s RBCs several times with saline
• Reverse typing- saline replacement technique
- serum is removed and replaced by an equal volume of saline
*True agglutination- RBC clumping will still remain after the
addition of saline
 Resolution of Common Group III
Discrepancies
 Wharton’s jelly- viscous mucopolysaccharide
material present on cord blood cells that may cause
the red blood cells’ aggregation
• Resolution: Thoroughly washing cord cells six to eight
times with saline
 Group IV Discrepancies
• between forward and reverse groupings are due to
miscellaneous problems
- 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 of Common Group IV
Discrepancies
 Cold reactive autoantibodies
- cause spontaneous agglutination of the patient’s cells
- often yield a positive direct Coombs’ or antiglobulin
test
- for example, anti-I antibody in the serum reacts with
all adult cells, as well as the reagent A1 and B cells
 Resolution of Common Group IV
Discrepancies
• Resolution:
1. Incubate patient’s RBC at 37°C for a short period, then
washed with saline at 37°C three times and retyped
2. RBCs can be treated with 0.01 M dithiothreitol (DTT) to
disperse IgM-related agglutination
3. reagent RBCs and serum can be warmed to 37°C for 10 to
15 minutes, mixed, tested, and read at 37°C
 Resolution of Common Group IV
Discrepancies

 Unexpected ABO isoagglutinins

• Antibodies on patient’s serum react at RT with the
corresponding antigen present on the reagent cells
- A2 and A2B individuals, who can produce naturally occurring
anti-A1
- A1 and A1B individuals, who may produce naturally
occurring anti-H
 Resolution of Common Group IV
Discrepancies
• Resolution:
- Serum grouping can be repeated using at least three
examples of A1, A2, B cells; O cells; and an autologous control
- patient’s RBCs can be tested with Dolichos biflorus to
confirm the presence of the ABO subgroup
 Resolution of Common Group IV
Discrepancies
 Unexpected alloantibodies other than ABO isoagglutinins
(e.g., anti-M)
• Reagent cells possess other antigens in addition to A1 and B
• Resolution:
- antibody identification panel should be performed with the
patient’s serum
 Rare Group IV Discrepancies
• Antibodies other than anti-A and anti-B may react to form
antigen-antibody complexes that may then adsorb onto
patient’s RBCs
- antibody against acriflavine, the yellow dye used in some
commercial anti-B reagents
• Resolution: Washing the patient’s cells three times with
saline and then retyping them should resolve this
discrepancy
 Rare Group IV Discrepancies
 Cis-AB refers to the inheritance of both AB genes from one
parent carried on one chromosome and an O gene inherited
from the other parent
• this phenotype was first discovered in 1964 in a Polish family
• express a weakly reactive A antigen (analogous to A2 cells) and a
weak B antigen (mixed-field; typical of subgroup B3)
• Weak anti- reacts with all ordinary B RBCs, yet not with cis-AB
RBCs
• A and B transferase levels are lower than those found in
ordinary group AB sera
 Various hypotheses have been offered to
explain the cis-AB phenotype
1. unequal crossing over between the A and B gene with gene
fusion and the formation of a new gene
2. point mutation at the ABO locus, and an enzyme was
produced that was capable of transferring both A-specific
and B-specific sugars to the precursor molecule

*high incidence of cis-AB being found in Japan

Causes of ABO
Forward (RBCs) Missing/weak - A or B subgroup
Discrepancies
Ags - Disease (leukemia)
Extra Ags - Acquired B
- Rouleaux
- Cold-autoAbs
- Wharton’s jelly
Mixed field - Group O transfusion
- Stem cell trannsplant
- A3 or B3 phenotype

Reverse Missing/Weak - Newborns, Elderly,

(Plasma) Abs Immunocompromised

Extra Abs - Anti-A1

- Cold alloAbs
- Rouleaux
- Cold autoAbs
- Passively acquired Abs
(i.e. plasma exchange,
mismatched plts.)
 Rh
• refers to a specific red blood cell (RBC) antigen (D)
• a complex blood group system currently composed of 61
different antigenic specificities
• Second in importance only to ABO blood group system in
terms of transfusion
• Antigens are very immunogenic
• Antibodies are produced after exposure to foreign red
blood cells that may cause significant HDFN and HTRs
 History
 1939: Levine and Stetson described a hemolytic transfusion
reaction in an obstetrical patient
- It was postulated that the fetus and the father possessed a common
factor that the mother lacked; The isolated antibody specificity was
not identified, but reacted with 80% of cells tested
 1940: Landsteiner and Wiener described an antibody made by
guinea pigs and rabbits when they were transfused with rhesus
macaque monkey RBCs
- antibody agglutinated 85% of human RBCs and was named anti-Rh
- The name Rh was retained for the human-produced
antibody
- anti-rhesus formed by the animals was renamed anti-LW
 mid-1940s: five antigens were defined in the Rh system
 1980s: molecular testing further defined the structure of
the RH genes
Terminology
 Terminology
 Two are based on postulated genetic theories of Rh
inheritance
 Third describes only the presence or absence of a given
antigen
 Fourth was established by the International Society of
Blood Transfusion (ISBT) Committee on Terminology for Red
Cell Surface Antigens

*Genotype vs. Phenotype is applied here

 1. Fisher-Race: DCE Terminology
• Fisher and Race defined the
five common Rh antigens
• postulated that the antigens
of the system were produced
by three closely linked genes
(D/d, C/c, and E/e)
• Antigens of the system were
named D, d, C, c, E, and e
• Now: There is no “d” antigen
 Fisher-Race theory (mid-1940s)
• individual inherits a set of RH genes from each parent called
haplotype
• pairing of maternal and paternal haplotypes determines the
offspring’s genotype (i.e., DCe/Dce)
Genotype written as 2 haplotypes separated by a / (i.e.,
DCe/Dce)
Deletions of specific genes is represented with a dash (i.e., D-)
Weakened antigen expression- placing parenthesis around (D),
(C), and (e)
Serologic
reactions to
Fisher-Race
nomenclature
 2. Wiener: Rh-Hr Terminology
• One gene responsible for
defining Rh that produced
an agglutinogen containing
three Rh factors
Agglutinogen= expressed
haplotype
Factors= antigens recognized
by an antibody
 Original Wiener Modified Wiener
Nomenclature Nomenclature
• named the five • R = presence of D antigen
common Rh antigens
as:
• r = absence of D antigen
 Rho = D • 1 or (‘) = presence of C
 rh’ = C • 2 or (“) = presence of E
 rh” = c • No 1 or (‘) = presence of c
 hr’ = E • No 2 or (“) = presence of e
 hr“ = e
 3. Rosenfield and Coworkers: Alphanumeric
Terminology (early 1960s)
• proposed a system that assigned a • Rh1 = D
number to each antigen of the Rh • Rh2 = C
system in order of its discovery or • Rh3 = E
recognized relationship to the Rh • Rh4 = c
system
• Rh5 = e
• A minus sign preceding a number Examples:
designates the absence of the
antigen  D+C+E+c-e-
- Rh: 1, 2, 3, –4, –5
• If an antigen has not been
phenotyped, its number will not  if e is not tested
appear in the sequence - Rh: 1, 2, 3, –4
 4. International Society of Blood Transfusion
Committee: Updated Numeric Terminology
• Establish a uniform nomenclature that is both eye- and
machine-readable and is in keeping with the genetic basis of
blood groups
• six-digit number for each authenticated antigen belonging to
a blood group system
- first three numbers: represent the system
- remaining three: antigenic specificity
 4. International Society of Blood Transfusion
Committee: Updated Numeric Terminology
• When referring to individual antigens = Rosenfield
nomenclature
 D + C – E + c + e + or DcE/ce or R2r  RH:1, –2, 3, 4, 5
Gene/allele/haplotype- symbols are italicized
Haplotype is followed by a space or an asterisk and numbers
are separated by commas (i.e., R1 haplotype or DCe would
be RH 1,2,5 or RH*1,2,5)
 Genetics
• RHD and RHCE - two closely linked genes located on chromosome 1
• RHD- codes for the presence or absence of the RhD protein
• RHCE- codes for either RhCe, RhcE, Rhce, or RhCE proteins
 Rh-Associated Glycoprotein (RHAG)
• RHAG- important gene for Rh antigen expression resides on
chromosome 6
• Rh-associated glycoprotein- very similar in structure to the
Rh proteins, but it is glycosylated
• termed a coexpressor
• mutations in the RHAG gene can result in missing or
significantly altered RhD and RhCE proteins
 Rh-Positive Phenotypes
 Normal Rh inheritance pattern:
• inherit one or two RHD genes
• two RHCE genes are inherited, one from each parent
*mutations in the RHD gene cause weakened expression of
the RhD antigen detected in routine testing and is generally
termed weak D
 Rh-Negative Phenotypes
• RBCs lack detectable D antigen
• arise from several pathways:
1. Complete deletion of the RHD gene- most common
2. RHD pseudogene or RHDψ- missense mutations in exon 5
& 6, a nonsense mutation in exon 6, and a 37-bp insertion
at the intron 3/exon 4 boundary
3. Altered RHD gene termed Del
 Most Probable Genotypes
• RH haplotypes is important to predict the correct most
probable genotype
example: Rh phenotype of D+C-E-c+e+
- Caucasian population the genotype Ror would be most likely
oRo haplotype = 4%
or haplotype = 37%
- African American population the genotype would be RoRo
oRo haplotype = 44%
RH Antigens
 Biochemistry
• Non-glycosylated proteins that reside on transmembrane proteins
and are an integral part of the RBC membrane
• RHD and RHCE are remarkably similar in that both encode for
proteins composed of 416 amino acids that traverse the cell
membrane 12 times
• Amino acid position 103 is important in determining C or c
expression
• position 226 differentiates E from e
• RhD and RhCE proteins and RhAG are exclusively on red blood cells
Hughes-Jones and
coworkers
measured the
number of D
antigen sites on a
variety of Rh
phenotypes
 Antigen Characteristics
• well developed at birth (can cause HDFN)
• Immunogenic
• The five common Rh antigens and their specific
corresponding antibodies account for the majority of
problems due to Rh antibodies
• D antigen does not have an allele
• C and c are alleles
• E is allelic to e
 D Antigen
• highly immunogenic
• the most potent
• Exposure to < 0.1 mL of Rh-positive RBCs can stimulate
antibody production in an Rh-negative person
• 85% of the general population is Rh-positive
• 15% are Rh-negative
• Anti-D was the most common cause of HDFN until Rh-
immune globulin was introduced
 Other Common Rh Antigens (C, E, c, e)
• c antigen - next most likely Rh antigen to elicit an immune
response, followed by E, C, and e
• inherited through the
RHCE gene
• alleles are codominant
 Weak D: Variations of D Antigen Expression
 Serologic weak D is noted when initial anti-D testing is negative or;
 ≤ 2+ strong, but detectable at the indirect antiglobulin testing (IAT)
phase
 Individuals with RBCs carrying weaker D antigen (historically called Du
type) can produce anti-D if they are missing epitopes of the D antigen
 Weak D types can be separated into three categories:
• position effect
• Quantitative
• partial-D antigen (missing one or more alleles)
* D epitopes on their RhCE protein
 1. Position Effect: C in Trans to D
• Or otherwise termed as gene interaction effect
• allele carrying RHD is trans to the allele carrying C (i.e., Dce/dCe)
• Rh antigen on the RBC is normal, but steric arrangement of the C
antigen in relationship to the D antigen appears to interfere with the
expression of D antigen
- does not occur when the C gene is inherited in the cis position to
RHD, such as DCe/dce
- D antigen is structurally complete
- These individuals can receive D-positive RBCs with no adverse
effects
 Weak D: Quantitative Changes Due to Fewer
D Antigen Sites
• D antigens expressed appear to be complete but fewer in number
• mutations in the RHD gene occur, causing changes or deletions in
amino acids present in the transmembrane or intracellular region of
the RhD protein
• single nucleotide polymorphisms (SNP) affects amino acid insertion
of the protein on the RBC membrane which results to altered RhD
antigen expression because there are fewer antigen sites overall
• rarely make anti-D
 Del
• phenotype occurring in individuals whose RBCs possess an
extremely low number of D antigen sites
• Adsorbing and eluting anti-D: often the only way to detect the D
antigen
o incubate anti-D with the RBCs in question at 37°C followed by
eluting the anti-D off the adsorbed red blood cells
• Southeast-Asian Del individuals have not been reported to make
anti-D
• Caucasian individuals- a few instances of anti-D production
• Sandler and others have suggested that patients who
initially type as D-negative should have RHD genotyping
performed to assess their true D status
 improve accuracy in Rh typing
conserve Rh-negative units for those who truly need them
prevent unnecessary administration of Rh immune
globulin in women with weak D serotypes
 Partial D or D Mosaic
• one or more D epitopes within the entire D protein is either
missing or altered
• Wiener and Unger postulated that the D antigen is made of
antigenic subparts (well-acceptrd theory)
• Tippett and Sanger worked with RBCs and sera of partial-D
individuals to classify these antigens. Seven categories were
recognized (I-VII)
• Category I is obsolete
• RHD gene being replaced by
Hybrid RhD-RhCe-RhD
portions of the RHCE gene
• These protein changes occur
external to the red blood cell
membrane
• Anti-D made by individuals
expressing partial D can cause
HDFN or transfusion reactions, or
both
• The identification of a person
with a partial D routinely occurs
after the person begins producing
anti-D
 Tippett and coworkers
• pursued the classification of partial-D antigens using
monoclonal anti-D (MAb-D)
• Today, a commercial monoclonal anti-D panel is available
• While partial D phenotypes are not common, category VI is
the most often encountered partial D phenotype
• A combination of serologic typing and molecular analysis is
often required to accurately categorize partial-D types
 D Epitopes on RhCE Protein
• RhCE protein can express RhD epitopes detected by some
monoclonal anti-D
• when typed with anti-D will show positive reactivity even
though the D epitope is on the RhCE protein
• Examples of these unusual phenotypes include DHAR and
ceCF (Crawford)
RH Antibodies
 Characteristics of Rh Antibodies
• most Rh antibodies are IgG immunoglobulins
• react optimally at 37°C or after antiglobulin testing
• Rh antibodies may show dosage, reacting preferentially with RBCs
possessing double-dose Rh antigen (3+ with E+e– RBCs versus 2+
with E+e+ RBCs)
• Antibodies are enhanced when testing with enzyme-treated RBCs
• IgG1, IgG2, IgG3, and IgG4 subclasses of Rh antibodies have been
reported.
• IgG1 and IgG3 are of the greatest clinical significance
• Rh antibodies often persist in the circulation for years
• low-titer Rh antibody may experience an anamnestic
(secondary) antibody response if exposed to the same
sensitizing antigen
• antigen-negative blood must be provided to any patient
with a history of Rh antibody sensitization
• Rh antibodies do not bind complement, therefore
intravascular, complement-mediated hemolysis does not
occur
• RBC destruction is primarily extravascular
 Rh Antibodies in Pregnancy
• primarily IgG and can traverse the placenta and may coat
fetal RBCs that carry the corresponding antigen
• positive direct antiglobulin test
• HDFN due to anti-D can be prevented with the use of Rh-
Immune Globulin
Testing for RH Antigens and
Antibodies
 Rh Typing Reagents
 The goal is to use a reagent anti-D that will allow for
typing individuals’ RBCs as quickly and accurately as typing
for ABO
• high-protein based
• low-protein based
• saline based
• chemically modified
• Monoclonal or blends of monoclonals
 Saline reactive reagents
• contain IgM immunoglobulin, were the first typing reagents
available to test for the D antigen
• low-protein based and can be used to test cells that are already
coated with IgG antibody (autoantibody)
• Primary disadvantages:
- limited availability
- cost of production
- lengthy incubation time
- it cannot be used for weak D typing (since it’s IgM)
 High-protein anti-D reagents
• consisted primarily of IgG anti-D (human plasma containing high-titer D-
specific antibody as raw material)
• Potentiators were added to optimize reactivity in the standard slide and rapid
tube tests
- bovine albumin
- macromolecular additives, such as dextran
• However, there is increase likelihood of false-positive reactions
• Major advantages
- incubation time
- ability to perform weak D testing and slide typing with the same reagent
- This type of anti-D reagent is also polyspecific
 Chemically modified Rh typing reagents
• alter the IgG anti-D molecule by breaking the disulfide bonds that
maintain the antibody’s rigid shape
• can be used for both slide and tube testing
• do not require a separate, manufactured Rh control as long as the
samples type as A, B, or O
• AB Rh-positive or when the Rh test is performed by itself, a separate
saline control or 6% to 8% albumin control must be used
• Fewer false-positive test reactions
• replaced the need for saline (IgM) anti-D reagents
 Rh monoclonal antibodies
• derived from single clones of antibody-producing cells that are
hybridized with myeloma cells to increase their reproduction rate
• usually a combination of monoclonal anti-D reagents to ensure
reactivity with a broad spectrum of Rh-positive RBCs
• Monoclonal blends can be used for slide, tube, microwell, and most
automated Rh testing
• monoclonal IgM anti-D reagents for column agglutination
techniques (FDA-approved)
• lack all potential for transmitting infectious disease
 Reagents for C, E, c, and e antigens
• aid in antibody identification and screening for antigen-
negative units of blood
• Rh antigen typing must be performed with:
- strict adherence to manufacturer’s directions
- use of proper controls
- accurate interpretation of test and control results
 Rh Testing Requirements for Pretransfusion,
Obstetric Patients, and Blood Donors
• AABB Standards- require that patients must be tested for the D
antigen, but additional testing for weak D is optional
- Delta check is performed
- All discrepancies must be fully resolved before the unit can be
labeled and released for transfusion
- Donors who test D-negative on the initial test must have additional
testing performed
- unit of blood is labeled as Rh-positive or Rh-negative
 Rh Testing Requirements for Pretransfusion,
Obstetric Patients, and Blood Donors
• patients are tested for the D antigen as part of routine
testing in combination with the ABO typing
• Rh-negative patients on the initial test are often interpreted
as Rh-negative and given Rh-negative units for transfusion
• Pregnant women are tested for the D antigen to see if they
are candidates for Rh-immune globulin
 Detecting and Identifying Rh Antibodies
• Patients who are pregnant or in need for transfusion must be tested for
the presence of all clinically significant antibodies, including the Rh
antibodies
• If Rh antibodies are suspected, then a complete antibody identification
workup should be performed
• Rh antibody reactivity can be enhanced by:
• Albumin
• low ionic strength solution (LISS)
• polyethylene glycol (PEG) to the test system
• treat the reagent red blood cells with proteolytic enzymes such as ficin
Selecting Compatible Blood for Transfusion
• Rh antibodies are clinically significant, so units that are
negative for the corresponding antigens must be selected for
transfusion
• Commercial antisera is available to test for the five common
Rh antigens, as well as some of the other less common types
• The need for blood negative for high-prevalence antigens can
be facilitated by contacting the blood supplier or the
American Red Cross American Rare Donor Program (ARDP)
Clinical Considerations
 Transfusion Reactions
• Circulating antibody can appear as soon as 10 to 14 days of a
primary exposure, and
• within 1 to 7 days after a secondary exposure
• extravascular destruction of immunoglobulin-coated RBCs
• recipient may have:
- unexplained fever
- mild bilirubin elevation
- decrease in hemoglobin and haptoglobin
- Positive direct antiglobulin test (DAT)
 Hemolytic Disease of the Fetus and Newborn
• HDFN caused by Rh antibodies is often severe because the
Rh antigens are well developed on fetal cells, and Rh
antibodies are primarily IgG, which readily cross the
placenta
• Rh-immune globulin- a purified preparation of IgG anti-D, is
given to D-negative woman during pregnancy and following
delivery of a D-positive fetus (w/in 72 hours)
• Passively acquired anti-D in the serum may be noted in
mothers who receive antepartum Rh-immune globulin
Rh Deficiency Syndrome: Rhnull
and Rhmod
 Rhnull syndrome
• fail to express any Rh antigens on the RBC surface
1. Amorphic- deletion of the RHD gene & mutation in each of the RHCE
genes
2. Regulator- mutation occurs in the RHAG gene
• negative for the high-prevalence antigen LW and for FY5
• Depressed S, s, and U antigens found on glycophorin B
• mild compensated hemolytic anemia, reticulocytosis, stomatocytosis, a
slight-to-moderate decrease in hemoglobin and hematocrit levels, an
increase in hemoglobin F, a decrease in serum haptoglobin, and possibly
an elevated bilirubin level
 Rhnull syndrome
• anti-Rh29 = potent antibody produced by Rhnull individuals exposed
to normal Rh cells
• individuals with Rhnull syndrome is necessary, only Rhnull blood can
be given
• The Rhnull phenotype is usually written in:
• Fisher-Race as —/—
• Wiener as Rhnull
• Rosenfield nomenclature as (RH: –1, –2, –3, –4, –5)
 Rhmod
• partial suppression of RH gene expression caused by
mutations in the RHAG gene causing weakened expression
of the normal Rh and LW antigens
• severely reduced expression of all Rh antigens
• S, s, and U antigen expression may be depressed
• exhibit RBC abnormalities similar to those with the Rhnull
syndrome; however, clinical symptoms are usually less
severe
Unusual Phenotypes and Rare
Alleles
 Cw

• was originally considered an allele at the C/c locus

• now known that the relationship between C/c and Cw is only
phenotypic
• antithetical to the high-prevalence antigen MAR
• single amino acid change most often found on the RhCe protein
• Anti-Cw: identified in individuals without known exposure to
foreign RBCs
- May show dosage
 f (ce)
• is expressed on the RBC when both c and e are present on the
same haplotype
• has been called a compound antigen, but is likely a single entity
resulting from conformational changes in the RHCE protein
• are expressed on the RHce protein
• Phenotypically, DCE/dce and DcE/DCe cells = D+C+E+c+e. However,
when tested with anti-f, only the DCE/dce shows positive reactivity
• Anti-f is generally a weakly reactive antibody reported to cause
HDFN and transfusion reactions
rhi (Ce)
• was considered a compound antigen present when C
and e are on the RhCe protein
• anti-rhi would only react with cells from an individual
with a haplotype of DCe or Ce
• Antigens cE (RH27) and CE (Rh22) also exist
 G
• present on most D-positive and all C-positive RBCs
• results from the amino acid serine at position 103 on the RhD,
RhCe, and RhCE proteins
• anti-G reacts as though it were a combination of anti-C plus anti-D
• G was originally described in an rr (ce/ce) person who received
D+C–E–c+e+ RBCs
- recipient produced an antibody that appeared to be antiD plus anti-
C, which should be impossible because the C antigen was not on the
transfused RBCs
 Anti-G
• Anti-G versus anti-D and anti-C is important when
evaluating obstetric patients
- if the patient has produced anti-G and not anti-D, then she
is considered a candidate for Rh-immune globulin
• For transfusion purposes, it is not necessary to discriminate
anti-D and anti-C from anti-G, as the patient would receive
D-negative and C-negative blood regardless if the antibody
is anti-D, anti-C, or anti-G
 Rh17 (Hr0)
• an antigen present on all RBCs with the “common” Rh
phenotypes (e.g., R1R1, R2R2, rr)
• antibody is directed to the entire protein resulting from the
RHCE genes
• When RBCs phenotype as D–, the most potent antibody
they make is often one directed against Rh17 (Hr0), which
would react with all cells except D–
 Rh23, Rh30, Rh40, and Rh52
• Rh23, Rh30, Rh40 , and Rh52 are all low-prevalence antigens
associated with a specific category of partial D

 Rh30 (also known as Goa or Dcor) is a marker for partial-D IVa

 Rh23 (also known as Wiel and Dw) is an antigenic marker for
category Va partial-D
 Rh52 or BARC is associated with some partial-D VI types
 Rh40 (also known as Tar or Targett) is a marker for partial-D VII
 Rh33 (Har)
• low-prevalence antigen associated with the rare variant haplotype
called R0Har
• codes for:
- normal amounts of c
- reduced amounts of e, f, Hr0
- reduced amounts of D antigen written as (D)c(e)
• D reactions are frequently so weak often typed as Rh-negative
• results from a hybrid gene RHCE-RHD-RHCE in which only a small
portion of RHD is inserted into the RHCE gene
Rh43 (Crawford)
• low-prevalence antigen on a variant Rhce protein
• results from a specific amino acid change in the RHce
gene, resulting in an RhD epitope on the Rhce protein
Rh32
• low-prevalence antigen associated with a variant of
the R1[D(C)(e)] haplotype called “R double bar N”
• C antigen and e antigen are expressed weakly
• D antigen expression is exaggerated or exalted
 e Variants
• individual may have a phenotype of e-positive but produce antibodies
behaving as anti-e
• result from multiple mutations in the RHCE gene
• Rh antigens hrB (Rh31) and hrS (Rh19 )
- Normally present in individuals who possess normal RhCe or Rhce
protein
- lacking in individuals with normal RhcE or RhCE proteins
- Are lacking on Rhce proteins encoded by some variant RHCE genes
- When immunized, they may produce anti-hrB or anti-hrS
- generally nonreactive with e-negative red blood cells (appearing to have

anti-e-like specificity)
RHD-CE-D hybrid gene
• the variant RHCE gene associated with the hrB-
negative phenotype is usually found with RHD-CE-D
hybrid gene
• The resulting protein lacks D antigen but possesses an
unusual form of the C antigen
• The haplotype that includes the RHDIIIa-RHCE-DIIIa
hybrid gene and variant RHCE genes is referred to as
r’s[(C)ces]
 V and VS
• V- historically referred to as ceS
- It is most often found in individuals with Gly263 amino acid change
in Rhce;
• VS- also known as eS
- results from a single amino acid change that occurs at position
Val245 in the Rhce protein
- Most hrB individuals with r’S genotype are VS+. The Val245
mutation in this haplotype is found in the hybrid
• Most V+ individuals are also VS+
 Landsteiner Wiener Blood Group System
• Phenotypically, there is a similarity between the Rh and LW systems
• Anti-LW reacts:
- strongly with most D-positive RBCs
- weakly with Rh-negative RBCs (and sometimes not at all)
- never with Rhnull cells
• Anti-LW shows equal reactivity with cord cells regardless of their D
type
• more frequently appears as an autoantibody
 Anti-LW
• special techniques can help distinguish between Anti-D and LW
antibodies
1. Treat the reagent panel cells with 0.2 m dithiothreitol (DTT) and
test the patient serum against the treated cells
- LW antigens are denatured with DTT treatment while D antigens are
unaffected
2. test the patient serum against Rh-positive and Rh-negative cord
blood cells
- Anti-D will react only with the Rh-positive cord cells whereas the LW
antibodies will react with all cord cells tested, regardless of Rh type
 Blood Group Terminology
and Common Blood Groups
Lewis, P, I, MNS, Kell, Duffy, Kidd, and Lutheran
• currently 346 RBC antigens in 36 systems that are
formally recognized internationally
• carbohydrates (sugars) attached to glycoprotein or
glycolipid structures
- Lewis, P, and I
• amino acids on a protein
- MNS, Duffy, Kell, Kidd, and Lutheran
Blood Groups and Terminology
 Blood group system
 consists of:
- one or more antigens controlled at a single gene locus
- two or more very closely linked homologous genes with little or no
observable recombination between them
• Most blood group alleles are codominant
• Some genes code for complex structures that carry more than one
antigen
• Amorphic (silent) alleles exist that make no antigen
• null phenotype- both amorphic alleles are inherited
• regulator or modifying genes- alter antigen expression
• certain conventions are followed when writing alleles, antigens, and
phenotypes
- Genes are written in italics or underlined and their allele number or letter is
always superscript
- Antigen names are written in regular type without italics or underlining; some
antigens have numbers or superscript letters
1. M+ and K–
2. Fy(a+) and Jk(a–)
3. Fy(a–b+)
4. Sc:–1,2
5. S+s+; K–; Fy(a+b–)
- Antigens can also be written using the system symbol followed by the antigen
number (i.e., KEL1)
Numeric Terminology
• Each antigen is given a six-digit identification number
- The first three digits represent the system, collection,
or series
- the second three digits identify the antigen
Example: K antigen is 006001
Collections
• are antigens that have a biochemical, serologic, or
genetic relationship but do not meet the criteria for a
system
• assigned a 200 number
• Some of the previously established collections have
been established as a system, some have incorporated
antigens into an existing system
 All remaining RBC antigens are catalouged into:
• 700 series of low prevalence antigens
- less than 1%

• 901 series of high-prevalence antigens

- more than 90%

*previously termed as high- and low-frequency, and high-

and low-incidence
The Lewis System
ISBT: 007
Symbol: LE
• Lewis antigens are not intrinsic to
RBCs but are on type 1
glycosphingolipids that are passively
adsorbed onto the RBC membrane
from the plasma
• The antibody, later called anti-Lea
• An antibody, later called anti-Leb was
found that reacted with Le(a–)
individuals
• Lea and Leb antigens result from the
interaction of two fucosyltransferases
encoded by independent genes, Le,
FUT3 (Le/le) and Se, FUT2 (Se/se)
found on chromosome 19p13.3 and
19q13.3, respectively
Development of Lewis Antigens
• Lewis antigens produced in saliva and other secretions are
glycoproteins
• Lewis cell-bound antigens absorbed from plasma onto the
RBC membranes are glycolipids
- GI tract is thought to be the primary source of Lewis
glycolipid in plasma
• Lewis glycolipids are not detectable in the plasma until about
10 days after birth
• Individuals who inherit both Le and Se genes:
Le(a–b–) phenotype at birth  Le(a+b–) after 10 days 
Le(a+b+)  Le(a–b+)
• true Lewis phenotype, after about 6 years
Lewis Antigens & Antibodies
• Ags resistant to:
- Enzymes ficin and papain
- dithiothreitol (DTT)
- glycineacid EDTA

• Ab reactivity
- testing with enzyme-treated RBCs
 Other Lewis Antigens & their Antibodies
• Leab
- previously known as Lex
- present on all Le(a+b–) and Le(a–b+) RBCs and on 90% of cord RBCs
• Anti-Leab
- fairly common and is frequently found with anti-Lea or anti-Leb
- heterogeneous and occurs mainly in Le(a–b–) secretors of group A1,
B, or A1B
• ALeb and BLeb - result from the addition of the A or B
immunodominant sugar, respectively, to type 1H
 Lewis Antibodies
• naturally occurring IgM and made by Le(a–b–) persons
• can bind complement
• do not cause hemolytic disease of the fetus and newborn (HDFN)
• Anti-Lea and anti-Leb can be neutralized by the Lewis substances
present in plasma or saliva
• Antibodies agglutinate saline-suspended RBCs, but these
agglutinates are often fragile
• Anti-Lea - most commonly encountered of the Lewis antibodies
- detected in RT tests, but it sometimes reacts at 37°C and in the IAT
 Lewis Antibodies
• anti-Leb
- not as common or generally as strong as antiLea
- usually an IgM agglutinin and can bind complement
- infrequently made by Le(a+b–)
1. anti-LebH - reacts best when both the Leb and the H
antigens are present on the RBC (i.e., O A2 cells)
2. anti-LebL - recognizes any Leb antigen regardless of the
ABO type
The P Blood Group
P1PK (003)
Globoside (028)
Related (209) Antigens
P Blood Group
 P1PK blood group system (ISBT 003) [22q13.2]
- P1, Pk, and NOR antigens
 Globoside blood group system (ISBT 028) [3q25]
- P and PX2 antigens
 Globoside collection (ISBT 209)
- LKE antigen
• 1927: Landsteiner and Levine; they injected rabbits with
human RBCs and produced an antibody, initially called anti-P,
that divided human RBCs into two groups: P+ and P–
• 1951: Levine and colleagues; described anti-Tja (now known
as anti-PP1Pk), an antibody to a high-prevalence antigen that
Sanger later showed was related to the P blood group
oAnti-P became anti-P1
oP+ phenotype became P1
oP– phenotype became P2
oP null individual became p
• Pk – described by Matson and coworkers in 1959 made P
blood group more complex
• When RBCs are tested only with anti-P1 and not with anti-P, the phenotype
should be written as P1+ (or P1) or P1–
• Only when P1– RBCs are tested and found to be reactive with anti-P should they
be designated as phenotype P2
 The antibodies:
- clinically insignificant
- potently hemolytic
- Reactivity of the antibodies can be greatly enhanced by
testing with enzyme-treated RBCs
 The antigens:
- synthesized by sequential action of glycosyltransferases,
which add sugars to precursor substances
- precursor of P1 can also be glycosylated to type 2H chains
- exist as glycosphingolipids
- resistant to treatment with ficin and papain, DTT,
chloroquine, and glycine-acid EDTA
Genetics
 The P1 Antigen
• poorly expressed at birth
• may take up to 7 years to be fully expressed
• Antigen strength in adults varies from one individual to another
- P1 strong (P1+s) and P1 weak (P1+w)
- vary with race
- In(lu) phenotype Lu(a–b–)
• deteriorates rapidly on storage
• When older RBCs are typed or used as controls for typing reagents
or when older RBCs are used to detect anti-P1 in serum, false-
negative reactions may result
 Anti-P1
• common, naturally occurring IgM antibody in the sera of P1–
individuals (rare IgG)
• weak, coldreactive saline agglutinin optimally reactive at 4°C
• can be neutralized or inhibited with soluble P1 substance
• anti-P1 that react only at temperatures below 37°C can be
considered clinically insignificant
• Rare examples of anti-P1 that react at 37°C can cause in vivo
RBC destruction; both immediate and delayed HTRs have
been reported
Other Sources of P1 Antigen and Antibody
• Discovery of strong anti-P1 in two P1– individuals
infected with Echinococcus granulosus tapeworms led
to the identification of P1 and Pk substance in hydatid
cyst fluid
• Also been found in patients with fascioliasis (bovine
liver fluke disease) and in bird handlers
• Soluble P1 substances can be used to neutralize anti-
P1
 Anti-PP1Pk

• Originally called anti-Tja; first described in the serum of Mrs. Jay, a p

individual with adenocarcinoma of the stomach
• produced by p individuals early in life without RBC sensitization and
reacts with all RBCs except those of the p phenotype
• separable through adsorption (anti-P, anti-P1, and antiPk)
• IgM and IgG type, react over a wide thermal range and efficiently
bind complement, which makes them potent hemolysins
• Anti-PP1Pk has the potential to cause severe HTRs and HDFN
• associated with an increased incidence of spontaneous abortions in
early pregnancy
Alloanti-P
• naturally occurring alloantibody in the sera of Pk
individuals
• potent hemolysin reacting with all cells except the
autocontrol and those with the p phenotype
• Alloanti-P is rarely seen, but it is very significant in
transfusion.
• IgG class alloanti-P may occur and has been
associated with habitual early abortion
 Autoanti-P Associated With Paroxysmal Cold
Hemoglobinuria (PCH)
• associated with the cold-reactive IgG autoantibody in patients with
paroxysmal cold hemoglobinuria (PCH)
• the IgG autoantibody in PCH is described as a biphasic hemolysin
- in vitro, the antibody binds to RBCs in the cold, and, via
complement activation, the coated RBCs lyse as they are warmed to
37°C
• demonstrable only by the Donath-Landsteiner test
• Also known as Donath-Landsteiner biphasic autoantibody
Antibodies to Compound Antigens
• Most examples are cold-reactive agglutinins
• antibodies requiring more than one antigenic
determinant have been described, including anti-IP1, -
iP1, -ITP1, and –IP
 Luke (LKE) Antigen
• 1965, Tippett and colleagues described an antibody in the
serum of a patient with Hodgkin’s lymphoma
- 84% tested Luke+
- 14% were weakly positive or Luke(w)
- 2% were Luke–
• associated with metastasis in renal cell carcinoma
 Luke (LKE) Antigen
• frequency of 98% in all populations and is expressed on cord
cells
• Decreased expression of the LKE- in individuals with the Se
gene having a 3-4-fold decreased risk of E.coli infections
• All individuals with the p and Pk phenotype are Luke–
 PX2 Antigen
• product of B3GALNT1 gene of GLOB blood group system
(028)
• has a frequency of >99.9% in all population and is expressed
on cord cells
• Enzyme treatment enhances the expression of the PX2
antigen
• Antibodies appear to be naturally occurring and primarily
IgG with a possible mixture of IgM that react best at IAT on
papain-treated RBCs
 Disease Associations
P BLOOD GROUP DISEASE/S ASSOCAITED
AG or AB
Anti-P1 Parasitic infections (E. granulosus, fasciolosis)
Anti-PP1Pk & Anti-P spontaneous abortions/Early abortions
Autoanti-P PCH
P system antigens receptors for P-fimbriated uropathogenic E. coli
Pk receptor for Shiga toxins
P receptor of human parvovirus B19
The I (027) System and i Antigen
• 1956: Wiener and coworkers gave a name to one cold agglutinin in
the serum of normal individuals and in patients with acquired
hemolytic anemia calling its antigen I for “individuality
• ISBT: correct designation is “I adult and i adult”
• I and i are not antithetical antigens
• branched and linear carbohydrate structures formed by the action
of glycosyl transferases
• I has been raised to blood group system status (system number
027, symbol I)
• i antigen remains in the Ii collection (collection number 207,
symbol I)
 The I and i Antigens
• I and i are high-prevalence antigens, but they are expressed in a
reciprocal relationship
• At birth, infant RBCs are rich in i, while I is almost undetectable
• first 18 months of life, the quantity of i slowly decreases as I
increases until adult proportions are reached
• adult RBCs are rich in I and have only trace amounts of i antigen
• no true I– or i– phenotype
• Some appear not to change their i status after birth and they
become the rare i adult
• Treatment of RBCs with ficin and papain enhances reactivity of the I
and i antigens with their respective antibodies. The I and i antigens
are resistant to treatment with DTT and glycine-acid EDTA
 Anti-I
• is a common autoantibody that can be found in virtually all
sera
• weak, naturally occurring, saline-reactive IgM agglutinin
with a titer less than 64 at 4°C.
• Incubating tests in the cold enhances anti-I reactivity and
helps confirm its identity; albumin and enzyme methods
also enhance anti-I reactivity. Testing enzyme-treated RBCs
with slightly acidified serum may even promote hemolysis
 Anti-I
• Pathogenic autoanti-I consists of strong IgM agglutinins with
higher titer and a broad thermal range of activity, reacting
up to 30°C
• stimulated by microorganisms carrying I-like antigen on their
surface (i.e., M. pneumoniae)
 Alloanti-I
• exists as an IgM or IgG antibody in the serum of most
individuals with the i adult phenotype
• strong autoanti-I can mimic alloanti-I: if enough
autoantibody and complement are bound to a patient’s
RBCs, blocking the antigenic sites, they may falsely type I-
negative
• not associated with HDFN because the antibody is IgM,
and the I antigen is poorly expressed on infant RBCs
 Anti-i
• Alloanti-i has never been described
• Autoanti-i is a fairly rare antibody that gives strong reactions with
cord RBCs and i adult RBCs and weaker reactions with I adult RBCs
• IgM and react best with salinesuspended cells at 4°C
• Potent examples are associated with infectious mononucleosis
(Epstein-Barr virus infections) and some lymphoproliferative
disorders
• they seldom cause significant hemolysis
• IgG anti-i has also been described and has been associated with
HDFN
 Biochemistry & Genetics

• I and i antigens are precursors for the synthesis of ABO and Lewis antigens
• ABH and Ii determinants on the RBC membrane are carried on type 2 chains
that attach either to proteins or to lipids
• i antigen is defined by at least two repeating N-acetyllactosamine units in
linear form
• The IGnT (also known as GCNT2) gene on chromosome 6p24.2 encodes the N-
acetylglucosaminyltransferase, which adds GlcNAc to form the branches
 The I T Antigen and Antibody
• 1965, Curtain and coworkers23 reported a cold agglutinin in
Melanesians that did not demonstrate classical I or i specificity
- The agglutinin reacted strongly with cord RBCs, weakly with
normal adult RBCs, and most weakly with i adult RBCs
• was frequently found in two populations: Melanesians and the
Yanomami Indians in Venezuela
• Examples of IgM and IgG anti-IT reacting preferentially at 37°C
have also been found in patients with warm autoimmune
hemolytic anemia, with a special association with Hodgkin’s
disease
Antibodies to Compound Antigens
• Bearing in mind the close relationship of I to the
biochemical structures of ABH, Lewis, and P antigens
• anti-IA, -IB, -IAB, -IH, -iH, -IP1, -ITP1, -IHLeb, and –iHLeb
• both antigens must be present on the RBCs for the
antibody to react
 Disease Associations
• Autoanti-I with cold agglutinin disease and M. pneumoniae
• Autoanti-i with infectious mononucleosis
• increased i antigen with shortened marrow maturation time or
dyserythropoiesis
• Hereditary erythroblastic multinuclearity with a positive acidified
serum test (HEMPAS)- much greater i activity on RBCs than control
cord RBCs
• In Asians, the i adult phenotype has been associated with
congenital cataracts
The MNS System
(002)
MNS
• anti-M and anti-N, both of which were reported in 1927
• Walsh and Montgomery discovered S in 1947
• s, the antithetical of S was discovered in 1951
• an antibody to a high-prevalence antigen, U (for almost
universal distribution), was named by Weiner In 1953
• Greenwalt and colleagues observed that all U– RBCs were also
S–s– resulted in the inclusion of U into the system
• The genes encoding the MNS antigens are located on
chromosome 4
 M and N Antigens
• found on a well-characterized glycoprotein called glycophorin
A (GPA)
- major RBC sialic acid–rich glycoprotein (sialoglycoprotein,
SGP)
• M and N antigens are antithetical and differ in their amino
acid residues at positions 1 and 5
- M: serine at position 1 and glycine at position 5
- N: leucine at position 1 and glutamic acid at position 5
 M and N Antigens
• The antigens are well developed at birth
- easily destroyed by the routine blood bank enzymes ficin,
papain, and bromelin and by the less common enzymes
trypsin and pronase, ZZAP, a combination of DTT and papain
or ficin
- they are not affected by DTT alone, 2-aminoethyliso-
thiouronium bromide (AET), α-chymotrypsin, chloroquine, or
glycineacid EDTA treatment
 S and s Antigens
• located on a smaller glycoprotein called glycophorin B (GPB) that is
very similar to GPA
- differentiated by the amino acid at position 29 on GPB. Methionine
defines S, whereas threonine defines s
• fewer copies of GPB (about 200,000) than GPA (106) copies per RBC
• S and s also are well developed at birth
• less easily degraded by enzymes
• Ficin, papain, bromelin, pronase, and α-chymotrypsin can destroy S
and s activity
 Anti-M
• naturally occurring saline agglutinins that react below 37°C
• 50% to 80% are IgG or have an IgG component
• do not bind complement, regardless of their immunoglobulin class, and they
do not react with enzyme-treated RBCs
• particularly common in patients with bacterial infections
• Shows dosage
• Antibody reactivity can be enhanced by increasing the serum-to-cell ratio or
incubation time, or both, by decreasing incubation temperature or by adding a
potentiating medium
• pH-dependent (best at pH 6.5), react only with RBCs exposed to glucose
solutions
 Anti-N
• cold-reactive IgM or IgG saline agglutinin that does not bind
complement or react with enzyme-treated RBCs
• demonstrate dosage
• potent anti-N can be made by the rare individual whose
RBCs type M+N–S–s–
• also seen in renal patients who were dialyzed on equipment
sterilized with formaldehyde regardless of MN type
- Dialysis-associated anti-N called anti-Nf
 Anti-S and Anti-s
• Most anti-S and anti-s are IgG, reactive at 37°C and the antiglobulin
phase of testing
• Demonstrate dosage effect
• may or may not react with enzyme-treated RBCs
• Although seen less often they are more likely to be clinically
significant because they may bind complement, and have been
implicated in severe HTRs with hemoglobinuria. They have also
caused HDFN.
• Units selected for transfusion must be antigen negative and
crossmatch compatible
 Genetics & Biochemistry
• The genes GYPA (M/N) and GYPB (S/s), which code for GPA and GPB,
respectively, are located on chromosome 4q31.21
• Alleles are codominant
• GYPA
- is considered to be the ancestral gene
- organized into seven exons
• GYPB
- has only five coding exons plus one noncoding or pseudoexon.
• GYPE
- it participates in gene rearrangements that result in variant alleles.
 GPA- and GPB-Deficient Phenotypes
• three rare phenotypes lack GPA or GPB or both GPA and GPB
 U– Phenotype
• U antigen is located on GPB very close to the RBC membrane
between amino acids 33 and 39
• lack GPB because of a partial or complete deletion of GYPB
• RBCs usually type S–s–U– and can make anti-U
• The U antigen is resistant to enzyme treatment
• Anti-U is typically IgG and has been reported to cause
severe and fatal HTRs and HDFN
• Some examples of anti-U weakly react with apparent U– by
adsorption and elution. Such RBCs are said to be U variant
(Uvar); these have an altered GPB that does not express S or
s
• Many examples of anti-U are actually anti-U plus anti-GPB
• If the patient is U– and N–, the antibody may actually be a
potent anti-N plus anti-U
 En(a–) Phenotype
• 1969: Darnborough and coworkers and Furuhjelm and colleagues
described an antibody to the same high-prevalence antigen, called Ena
(for envelope)
• En(a–) individuals appeared to be M–N– with reduced NeuNAc on
their RBCs
• En(a–) phenotype results from homozygosity for a rare gene deletion
at the GYPA locus; no GPA is produced, but GPB is not affected
- Anti-EnaTS- recognizes a trypsin-sensitive (TS) area on GPA
- anti-EnaFS reacts with a ficin-sensitive (FS)
- anti-EnaFR reacts with a ficin-resistant (FR)
• Anti-Ena has caused severe HTRs and HDFN
 Mk Phenotype
• 1964: rare silent gene Mk was named by Metaxas and Metaxas
Buhler when they found an allele that did not produce M or N
• A second family showed that Mk was also silent at the Ss locus
• 1979: two related MkMk blood donors were found in Japan
• RBCs of these individuals typed M–N–S–s–U–En(a–)Wr(a–b–), but
they had a normal hematologic picture
• Mk gene represents a single, near-complete deletion of both GYPA
and GYPB
• MkMk is the null phenotype in the MNS system
 Other Antibodies in the MNS System
 Antibodies to high-prevalence antigens
- easily detected with antibody detection RBCs
 Antibodies to lowprevalence antigens
- rarely detected by the antibody detection test but are seen as an
unexpected incompatible crossmatch or an unexplained case of HDFN
 Autoantibodies
- Not all examples of anti-M in M+ individuals or anti-N in N+ individuals
are autoantibodies
- Autoantibodies to U and Ena are more common and may be associated
with warm-type autoimmune hemolytic anemia
 Disease Associations
• MNS antigen can serve as receptors for complement,
bacteria, and viruses
• GPAM may serve as the receptor by which certain
pyelonephritogenic strains of E. coli
• GPA and GPB (also NeuNAc) used by malaria parasite
Plasmodium falciparum as alternative receptors
The Kell (006) and Kx (019)
Systems
• consists of 36 high-prevalence and low-prevalence antigens
• was the first blood group system discovered after the introduction of
antiglobulin testing
• 1946: Anti-K was identified in the serum of Mrs. Kelleher that reacted
with the RBCs of her newborn infant, her older daughter, her husband,
and about 7% of the random population
• 1949: anti-k, the high-prevalence antithetical partner to K, was
described
• 1957 and 1958: antithetical antigens Kpa and Kpb were described,
respectively
• 1958: Jsa and Jsb (described in 1963) were found to be antithetical and
related to the Kell system
• 1957: The discovery of the null phenotype, designated Ko
 K and k Antigens
• Kell blood group antigens are found only on RBCs
• K antigen can be detected on fetal RBCs as early as 10 weeks and is well
developed at birth
• k antigen has been detected at 7 weeks
• total number of K antigen sites per RBC is 3,500 up to 18,000 sites (very
immunogenic)
• rated second only to D in immunogenicity; appear to be induced by pregnancy
and transfusion
• The antigens are not denatured by enzymes ficin and papain
• destroyed by trypsin and chymotrypsin when used in combination. Thiol-
reducing agents, such as 100 to 200 mM DTT, 2-mercaptoethanol (2-ME), AET,
ZZAP (which contains DTT in addition to enzyme) and Glycine-acid EDTA (an
IgG-removal agent)
 a
Kp , b
Kp , and Kp c Antigens
• Kpa and Kpc are low-prevalence mutations of their
high-prevalence partner Kpb
• Kpa - s associated with suppression of other Kell
antigens on the same molecule, including k and Jsb
Js a and Jsb Antigens
• Jsa antigen, antithetical to the high-prevalence antigen
Jsb
• prevalence of Jsa in blacks is almost 10 times greater
than the prevalence of the K antigen in blacks
• The two antigens were linked to the Kell system when
it was discovered that Ko RBCs were Js(a–b–)
 Anti-K
• anti-K is the most common antibody seen in the blood bank (outside ABO and
Rh)
• IgG and reactive in the antiglobulin phase made in response to antigen
exposure
• Naturally occurring IgM examples of anti-K have been associated with
bacterial infections
- Marsh and colleagues33 studied an IgM anti-K in an untransfused 20-day-old
infant with an E. coli O125:B15 infection whose mother did not make anti-K
• has been implicated in severe HTRs and also associated with severe HDFN
• When a pregnant woman is identified as making anti-K, it is prudent to type
the father for the K antigen. If he is K+, the fetus should be monitored
carefully for signs of HDFN
 Antibodies to Kpa, Jsa, and Other Low-
Prevalence Kell Antigens
• rare because so few people are exposed to these antigens
• most often detected through unexpected incompatible
crossmatches or cases of HDFN
• serologic characteristics and clinical significance of these antibodies
parallel anti-K
• original anti-Kpa was naturally occurring, but most antibodies result
from transfusion or pregnancy
Antibodies to k, Kpb, Jsb, and Other High-
Prevalence Kell Antigens
• rare because so few people lack these antigens
• parallel anti-K in serologic characteristics and clinical
significance
• easy to detect but difficult to work with; reactivity
that is abolished with DTT or AET treatment suggests
that the antibody may be related to the Kell system
 Genetics & Biochemistry
• The gene KEL, located on chromosome 7q33, is organized into 19
exons of coding sequence
• Kell antigens are located on a glycoprotein that consists of 731
amino acids and spans the RBC membrane once
• Kell glycoprotein is covalently linked with another protein, called Xk,
and is dependent upon the presence of the Xk protein
- absence of Xk results in McLeod syndrome
• The gene XK, which encodes the Kx antigen, is independent of KEL
and is located on the short arm of the chromosome Xp21.1
The Kx Antigen
• Kx is present on all RBCs except those of the rare
McLeod phenotype
• Ko and Kmod phenotype RBCs have increased Kx
antigen
• When Kell antigens are denatured with AET or DTT,
the expression of Kx increases
 The Ko Phenotype and Anti-Ku
• Ko RBCs lack expression of all Kell antigens, but have no
membrane abnormality and survive normally in circulation
• Immunized individuals make an antibody called anti-Ku that
recognizes the “universal” Kell antigen (Ku) present on all
RBCs except Ko
• has caused both HDFN and HTRs
• can be made artificially by treating normal RBCs with DTT,
AET, or glycine-acid EDTA
 The McLeod Phenotype
• McLeod phenotype RBCs lack Kx and the Kell system high-
prevalence antigen, Km, and have marked depression of all other
Kell antigens
- has been associated with several mutations and deletions at the XK
locus
- significant proportion of the RBCs are acanthocytic with decreased
deformability and reduced in vivo survival. Individuals with the
McLeod phenotype have a chronic but often well-compensated
hemolytic anemia
 McLeod Syndrome
• one of the neuroacanthocytosis syndromes
• develop a slow, progressive form of muscular dystrophy between
ages 40 and 50 years and cardiomegaly
• associated neurological disorder presents initially as areflexia (a
lack of deep tendon reflexes) and progresses to choreiform
movements (well-coordinated but involuntary movements)
• also have elevated serum creatinine phosphokinase levels of the
MM type (cardiac/skeletal muscle) and carbonic anhydrase III levels
 McLeod and CGD
• McLeod males with CGD make anti-Kx + Km (sometimes called anti-
KL)
• Anti-Km is made by McLeod males without CGD
 Altered Expressions of Kell Antigens
• McLeod phenotype
• suppression by the Kpa gene
• rare Gerbich-negative phenotypes Ge: –2, –3, 4 and Ge: –2, –3, –4
 Kmod – umbrella term for other phenotypes with very weak Kell
expression
Patients with autoimmune hemolytic anemia, in which the
autoantibody is directed against a Kell antigen
K– patient who acquired a K-like antigen during a Streptococcus
faecium infection
Autoantibodies
• Mostly directed against undefined high-prevalence
Kell antigens
• autoantibodies to K, Kpb, and K13 have been reported
• Mimicking specificities have also been reported
The Duffy System
(008)
FY
• 1950: was named for Mr. Duffy, a multiply transfused
hemophiliac who was found to have the first described
example of anti-Fya
• 1951: its antithetical antigen, Fyb, was found in the serum of
a woman who had had three pregnancies
• The gene responsible for this null phenotype was called Fy
- FyFy appeared to be a common genotype in blacks,
especially in Africa
- Fy(a–b–) RBCs resist infection Plasmodium knowlesi and also
resist infection by Plasmodium vivax
 Fya and b
Fy Antigens
• can be identified on fetal RBCs as early as 6 weeks gestational age
and are well developed at birth
• Destroyed by enzymes ficin, papain, bromelin, and chymotrypsin,
and by ZZAP (which contains either papain or ficin in addition to
DTT)
• Resistant to DTT alone, AET, or glycine-acid EDTA treatment
• about 13,000 to 14,000 Fya or Fyb sites on Fy(a+b–) and Fy(a–b+)
RBCs, respectively
• Do not react with enzyme-treated RBCs
 Anti-Fya and Anti-Fy b

• Anti-Fya - a common antibody occurs three times less frequently

than anti-K
• Anti-Fyb - is 20 times less common than anti-Fya
• usually IgG and react best at the antiglobulin phase, rarely bind
complement
• Antibody activity is enhanced in a lowionic strength medium
• Some show dosage
• associated with acute and delayed HTRs
• Once the antibody is identified, Fy(a–) or Fy(b–) blood must be
given
 Genetics & Biochemistry
• gene ACKR1, formally known as DARC was linked to a visible, inherited
abnormality of chromosome 1
• located near the centromere on the long arm of chromosome 1q21-q22
• Fy locus is syntenic to the Rh locus
• three common alleles: Fya, Fyb, and Fy
• Fy gene in Fy(a–b–) blacks is predominantly an Fyb variant with a change
in the promoter region (GATA) of the gene
• Duffy antigens reside on a glycoprotein of 336 amino acids
- is a member of the superfamily of chemokine receptors and is known as
the atypical chemokine receptor 1
 Fy x

• described in 1965 as a new allele at the Fy locus

• is an inherited weak form of Fyb that reacts with some examples of
anti-Fyb
• may type Fy(b–), but their RBCs adsorb and elute anti-Fyb. They
also have depressed expression of their Fy3 and Fy5 antigens
• related to a reduced amount of Duffy glycoprotein on the surface
of RBCs
• There is no anti-Fyx
 Fy3 Antigen and Antibody
• 1971: anti-Fy3 was found in the serum of an Fy(a–b–) white
Australian female
• it was an inseparable anti-FyaFyb
• rare antibody made by Fy(a–b–) individuals who lack the Duffy
glycoprotein
- found in whites, Cree Indian families, and blacks
- Blacks with the Fy(a–b–) phenotype rarely make anti-Fy3
• Antigen is not destroyed by enzymes
 Fy5 Antigen and Antibody
• 1973, Colledge and coworkers42 discovered anti-Fy5 in the serum
of an Fy(a–b–) black child who later died of leukemia
• Sometimes, sera containing anti-Fy5 also contain anti-Fya
• have been reported in multiply transfused Fy(a–b–) patients with
sickle cell disease
• appears to be the result of interaction between the Rh complex
and the Duffy glycoprotein
• is not destroyed by enzymes
The Kidd System
(009)
JK
• simple and straightforward system consisting of only three
antigens
• 1951, Allen and colleagues reported finding an antibody in
the serum of Mrs. Kidd, whose infant had HDFN
• anti-Jka (for the infant John Kidd) reacted with 77% of
Bostonians
• antithetical partner, Jkb, was found 2 years later
• 1959: null phenotype Jk(a–b–) was described
• high-prevalence antigen called Jk3, which is present on any
RBC positive for Jka or Jkb was discovered due to antibody
produced against it
 Jka and Jk b Antigens
• commonly found on RBCs of most individuals
• are well developed on the RBCs of neonates
- Jka has been detected on fetal RBCs as early as 11 weeks
- Jkb has been detected at 7 weeks
• Kidd antigens are not very immunogenic
• antigens are not denatured by papain or ficin, and also not
affected by chloroquine, DTT, AET, or glycine-acid EDTA
 Anti-Jka and Anti-Jkb
• have a notorious reputation in the blood bank
• demonstrate dosage, are often weak, and are found in combination with
other antibodies
• Anti-Jka is more frequently encountered than anti-Jkb
• usually IgG (antiglobulin reactive) but may also be partly IgM and are made in
response to pregnancy or transfusion
• The titer of anti-Jka or anti-Jkb quickly declines in vivo
• Associated with delayed-type HTR, but rarely with HDFN
• treatment of RBCs with enzymes, LISS or PEG (to promote IgG attachment),
by using four drops of serum instead of two in a saline tube method generally
enhances antibody reactivity
 Genetics & Biochemistry
• The Jk locus is on chromosome 18q11-q12
• The gene SLC14A1 is a member of the urea transporter gene family
• silent Jk allele can arise from mutations in both the Jka and Jkb
alleles
• Jka and Jkb are inherited as codominant alleles
• 1982: Heaton and McLoughlin reported that Jk(a–b–) RBCs resist
lysis in 2M urea
• With Jk(a+) or Jk(b+) RBCs, lysis in 2M urea occurs within 1 minute;
with Jk(a–b–) cells, lysis is delayed by 30 minutes
Jk(a–b–) Phenotype and the Recessive Allele,
Jk
• Null Jk(a–b–) phenotype lacks all Kidd antigens;
although very rare, but is most abundant among
Polynesians
• No clinical abnormalities have been associated with
the Jk(a–b–)
• most Jk(a–b–) nulls are homozygous for the rare
“silent” allele Jk
 Jk(a–b–) Phenotype and the Dominant In(Jk)
Allele
• dominant gene called In(Jk) for “inhibitor”
• Dominant type Jk(a–b–) RBCs adsorb and elute anti-Jk3 and anti-
Jka or anti-Jkb
• indicating that the antigens are expressed but only very weakly
• dominant type Jk(a–b–) phenotype do not make anti-Jk3
• In(Jk) gene does not reside at the Jk locus and molecular basis is
unknown
Anti-Jk3
• Alloanti-Jk3 is an IgG antiglobulin-reactive antibody
that looks like an inseparable anti-JkaJkb
• reacts optimally by an antiglobulin test, and the
reactivity is enhanced with enzyme pretreatment of
the RBCs
• associated with severe immediate and delayed HTRs
and with mild HDFN
The Lutheran System
(005)
LU
• 1945: anti-Lua was found in the serum of a patient with lupus
erythematosus, following the transfusion of a unit of blood carrying the
corresponding low-prevalence antigen
• Antibody was named Lutheran for the donor; the donor’s last name was
Lutteran but the donor’s blood sample was incorrectly labeled
• 1956: Cutbush and Chanarin described anti-Lub, which defined the
antithetical partner to Lua
• 1961: Crawford and colleagues described the first Lu(a–b–) phenotype
(dominant type)
• 1963: Lu(a–b–) phenotype inherited as a recessive silent allele was
described
• Lutheran glycoprotein is widely distributed in tissues: especially fetal
hepatic epithelial cells
 Lua and Lub Antigens
• produced by allelic codominant genes
• Most individuals are Lu(b+)
• number of Lub sites per RBC is low, estimated to be from 1,640 to
4,070 on Lu(a–b+) RBCs and from 850 to 1,820 on Lu(a+b+) RBCs
• detected on fetal RBCs as early as 10 to 12 weeks of gestation, they
are poorly developed at birth
• antigens are resistant to the enzymes ficin and papain and to
glycine-acid EDTA treatment
• destroyed by treatment with the enzymes trypsin and α-
chymotrypsin.
 Anti-Lu a

• Most examples are IgM naturally occurring saline agglutinins that

react better at room temperature; some are capable of binding
complement
• more likely encountered as an incompatible crossmatch or during
an antibody workup for another specificity
• characteristic loose, mixed-field reactivity in a test tube
• no documented cases of immediate HTRs; there are only rare and
mild delayed HTRs
 Anti-Lu b

• most examples of anti-Lub are IgG and reactive at 37°C at

the antiglobulin phase
- made in response to pregnancy or transfusion
• Anti-Lub has been implicated with shortened survival of
transfused cells and post-transfusion jaundice, but severe
or acute hemolysis has not been reported
 Genetics & Biochemistry
• The LU gene BCAM is located on chromosome 19q13.2
- along with other genes (H, Se, Le, LW, Oka)
• antigens are located on a type 1 transmembrane protein
• Lutheran glycoproteins belong to the immunoglobulin superfamily
of proteins; homologous to immunoglobulin variable or constant
domains
• Lutheran proteins are multifunctional adhesion molecules that
bind laminin, notably in sickle cell disease
 Lu(a–b–) Phenotypes
• Three genetic explanations for the Lu(a–b–) phenotype have been described
 Dominant Type Lu(a–b–)
 Recessive Type Lu(a–b–)
 Recessive X-Linked Inhibitor Type
Anti-Lu3
• a rare antibody that reacts with all RBCs except Lu(a–
b–) RBCs
• recognizes a common antigen, Lu3, that is present
whenever Lua or Lub
• usually antiglobulin reactive. This antibody is made
only by individuals with the recessive type of Lu(a–b–)
The Diego System
(010)
DI
 Diego System
• composed of 22 antigens
• three sets of high and low pairs of antithetical antigens (Dia/Dib, Wra/Wrb, and
Wu/DISK) and 16 low-prevalence antigens
• was named after the first antibody maker in a Venezuelan family during an
investigation of HDFN
- anti-Dia had caused HDFN in a Venezuelan baby
• antigens are carried on Band 3, also known as the red blood cell anion
exchanger (AE1) or solute carrier family-4 anion exchanger, member 1
(SLC4A1)
• Gene SLC4A1, consist of 20 exons and is located on chromosome 17q21.31
• The Di(a–b–) phenotype has not been reported
• Wra and Wrb are associated with an amino acid substitution
on the fourth external loop, close to the insertion point of
the protein into the RBC membrane
• expression of Wrb requires the interaction of band 3 and
normal GPA of the MNS blood group system. GPA-deficient
RBCs are Wr(a–b–)
• Diego antigens are expressed on RBCs of newborns
• antigens are resistant to treatment with ficin and papain,
DTT, and glycine-acid EDTA, with the exception of Bpa, which
is sensitive to papain
 Diego Antibodies
• Diego system antibodies are sometimes IgM, but are usually IgG,
reactive in the antiglobulin phase of testing
• anti-Dia and anti-Dib have caused hemolytic transfusion reactions
(HTRs) and HDFN
• Anti-Wra is a relatively common antibody in donors and patients;
some are directly agglutinating, but most require the antiglobulin
phase of testing to be detected and has caused severe HTRs
• anti-ELO, which has caused severe HDFN
• Autoanti-Wrb is relatively common in the serum of patients with
warm autoimmune hemolytic anemia
The Yt System
(011)
Yt
• was named in 1956 for the first antibody maker and used the last letter
“t” in the patient’s last name, which was Cartwright. Apparently “why T”
became “Yt”
• Yta is the high-prevalence antigen in all populations
• Ytb is the low-prevalence antigen
• Three phenotypes are observed:
- the common Yt(a+b–) and Yt(a+b+) and the rare Yt(a–b+)
- The Yt(a–b–) phenotype has not been reported
• Yt antigens are antithetical and represent an amino acid substitution on
the glycosylphosphatidylinositol (GPI)- linked RBC glycoprotein
acetylcholinesterase (AChE)
• The gene ACHE is located on chromosome 7 at position 7q22
• Yt antigens
- variably sensitive to ficin and papain, are sensitive to DTT
- resistant to glycine-acid EDTA
- are developed at birth but are expressed weaker on cord RBCs than on adult
RBCs
- absent from RBCs of people with paroxysmal nocturnal hemoglobinuria (PNH)
III
• Anti-Yta and anti-Ytb are IgG and are stimulated by pregnancy or transfusion
• Anti-Yta is reasonably immunogenic
• However, Ytb appears to be a poor immunogen, as the antibody is rare.
• Yt antibodies have not caused HDFN
• Monocyte phagocytosis assays (MMA) may be helpful in determining clinical
significance of anti-Yta
The Xg System
(012)
XG
• 1962: Anti-Xga was discovered in the serum of a multiply
transfused man
• antigen Xga expression was controlled by an X-linked gene
• The antigen was named after the X chromosome and g for “Grand
Rapids,” where the patient was treated
• Gene XG encoding Xga is located on the chromosome Xp22.32
• The gene MIC2, responsible for CD99 is located on the
chromosome Xp22.2 and Y chromosome at Yp11
• all Xg(a+) individuals have a high expression of CD99 on their RBCs,
and all Xg(a–) females have a low expression of CD99
• 68% of Xg(a–) males have a high expression and 32% have a low
expression of CD99
• Cord RBCs express Xga weakly
• The antigen is sensitive to ficin and papain but
resistant to DTT treatment
• Anti-Xga is usually IgG; some examples are naturally
occurring
- has not been implicated in HDFN or as a cause of HTRs
- Only a few examples of anti-CD99 have been reported
The Scianna System
(013)
SC
• currently consists of seven antigens
• named after the first antibody maker
• 1962: a new high-prevalence antigen was named Sm
• 1963: a new low-prevalence antigen, Bua was found
• Both are antithetical so they are renamed:
- Sm: Sc1
- Bua: Sc2
• 1980: an individual in the Marshall Islands in the South Pacific was
found with the Sc:–1,–2 phenotype and made an antibody to an
unknown high-prevalence antibody. The antibody was named anti-
Sc3
• Sc:–1,–2,–3 phenotype is the Scianna null type
• The SC gene ERMAP is located on chromosome 1p34.2
- product of the gene is a protein called erythroid membrane-associated
protein (ERMAP), which is an RBC adhesion protein
• The low-prevalence antigen Rd became Sc4;
• high-prevalence antigens Sc5 (STAR), Sc6 (SCER), and Sc7 (SCAN)
• Scianna antigens are resistant to ficin and papain and to DTT treatment,
except for Sc2, which has variable reactivity
• The antigens are expressed on cord RBCs
• Alloantibodies are usually IgG and reactive in the antiglobulin phase
• None have been reported to cause a severe HTR
• one severe case of HDFN for anti-Rd, for which the baby required exchange
transfusion
• Autoantibodies to Sc1 and Sc3 have been reported
The Dombrock System
(014)
DO
• was named for the first antibody maker, Mrs. Dombrock (anti-Doa),
found in 1965
• Anti-Dob, antithetical antigen Dob, was identified in 1973
• three resulting phenotypes
 Do(a+b–)
 Do(a+b+)
 Do(a–b+)
• 1967: high-prevalence antigens Gya and Hy were both described
• 1972: high-prevalence antigen Joa was described
• 1995: it was reported that Gy(a–) RBCs were also Do(a–b–)
• The Gy(a–) phenotype is the Dombrock null
• Dombrock antigens are carried on a mono-ADPribosyltransferase 4
(ART4) attached to the RBC membrane by a GPI anchor
• The gene ART4 encoding the Dombrock glycoprotein is located on
chromosome 12p13-p12
• Dombrock antigens are resistant to ficin, papain, and glycine-acid EDTA
and are sensitive to 0.2 M DTT treatment
• The antigens are present on cord RBCs, but are absent from PNH III RBCs
• The Doa and Dob antigens are poor immunogens
• Gya, however, is highly immunogenic
• Anti-Doa and anti-Dob have caused delayed HTRs but no clinical HDFN
- are usually IgG, reacting optimally with enzyme-treated RBCs; usually
weakly reactive and disappear
The Colton System
(015)
CO
• was named in 1967 for the first antibody maker; it should have
been named Calton, but the handwriting on the tube was misread!
• antithetical antigens are Coa (high-prevalence) and Cob (low-
prevalence)
• third antigen, Co3, is present on all RBCs except those of the very
rare Co(a–b–) phenotype
• Co4, a high-prevalence antigen, has been identified on RBCs from
two individuals with the Co(a–b–) phenotype
• Colton antigens are expressed on RBCs of newborns and are
resistant to treatment with ficin and papain, chloroquine, and DTT
• Colton antigens are carried on aquaporin 1 (AQP1), which accounts
for 80% of water reabsorption in the kidneys
• The gene AQP1 is located on chromosome 7p14
• Antibodies are usually IgG and are enhanced with enzyme-treated
RBCs
• Anti-Coa is has been reported to cause HTRs and HDFN
• Anti-Cob appears more often with other specificities but has also
caused HTRs and mild HDFN
• Anti-Co3, which reacts with all Co(a+) and Co(b+) RBCs, has been
reported to cause mild HTR and severe HDFN
The Landsteiner-Wiener System
(016)
LW
• had its origins along with the discovery of the D antigen of the Rh
blood group system
• There are three LW antigens: LWa, LWab, and LWb
• ISBT has used LW5, LW6, and LW7 as the antigen numbers
• LWab was originally defined by the antibody made by an individual
with an inherited LW(a–b–) phenotype (Mrs. Big)
• Rhnull RBCs also type LW(a–b–) and are considered to be the only
true LW– RBCs
• LW antigens is a glycoprotein known as intracellular adhesion
molecule 4 (ICAM-4), a member of the immunoglobulin
superfamily.
• The LW gene ICAM4 is located on chromosome 19p13.2
• Anti-LW usually reacts strongly with D+ RBCs, weakly (and sometimes
not at all) with D– RBCs from adults and not at all with Rhnull RBCs
• anti-LW reacts equally well with cord RBCs regardless of their D type
• Distinguishing anti-LW from anti-D is most easily accomplished by testing
DTT-treated D+ RBCs
• LW antigens are resistant to treatment of RBCs with enzymes and
glycine-acid EDTA
• LW antigens may be depressed during pregnancy and in some diseases,
such as lymphoma and leukemia
• Autoanti-LW is also common in serum from patients with warm
autoimmune hemolytic anemia
• No anti-LW has been shown to cause serious HDFN or transfusion
reactions
The Chido/Rodgers System
(017)
CH/RG
• was named after the first two antibody producers, Ch for Chido and
Rg for Rodgers, described in 1967 and 1976, respectively
• Serologically, both were characterized as nebulous
• Ch and Rg antigens are not intrinsic to the RBC membrane, but they
are on the complement protein (C4), and are adsorbed onto RBCs
from plasma
• C4 glycoprotein has two isoforms: C4B carries the Ch antigens, and
C4A expresses Rg antigens
• The genes C4A and C4B at two closely linked loci located on
chromosome 6p21.3
• consists of nine antigens: Ch1 to Ch6, Rg1, and Rg2 are all high
prevalence, and WH
• antigens are destroyed by ficin and papain but are resistant to
treatment with DTT and glycine-acid EDTA.
• Anti-Ch and anti-Rg are usually IgG and react weakly,
• Neutralization of anti-Ch and anti-Rg with pooled plasma is often
used as part of the identification of these antibodies in a patient’s
serum
• Anti-Ch and anti-Rg are clinically insignificant for transfusion
The Gerbich System
(020)
GE
• was named in 1960 after Mrs. Gerbich, the first antibody producer
• currently six high-prevalence Gerbich antigens (Ge2, Ge3, Ge4, GEPL,
GEAT, and GETI) and five low-prevalence antigens (Wb, Lsa, Ana, Dha, and
GEIS)
• antigens are carried on sialoglycoprotein structures GPC and GPD
• GPC and GPD are both encoded by the GYPC gene, located on
chromosome 2q14.3
• Three Gerbich-negative phenotypes:
- Ge:–2,3,4 (Yus type)
- Ge:–2,–3,4 (Gerbich type)
- Ge:–2,–3,–4 (Leach type) Gerbich null
* The RBCs of Ge null phenotype are elliptocytic and these individuals
may have a mild anemia
• Gerbich antigens are expressed at birth
• Gerbich or Leach phenotypes have weak expression of Kell blood
group antigens
• antigens are resistant to treatment with DTT and glycine-acid EDTA
• Ge2 and Ge4 are ficin and papain sensitive, but Ge3 is ficin resistant
• Gerbich antibodies may be IgM, but most are IgG and can be eluted
from DAT+ cord bloods, but only three cases of serious HDFN due to
anti-Ge3 have been reported, and two were children of the same
mother
• Anti-Ge2 is the most common of the Gerbich antibodies.2 It is the
antibody made by the Yus phenotype
• Anti-Ge4 is a very rare antibody
The Cromer System
(021)
CROM
• 1965, an antibody was found in a black prenatal patient, Mrs.
Cromer, that reacted with all RBCs except her own and two siblings
- The antibody was named anti-Cra in 1975
- Cromer antigens are carried on decay-accelerating factor (DAF,
CD55), a complement-regulatory protein
- The gene CD55 is located on chromosome 1q32
- PNH III RBCs are deficient in DAF so they also lack Cromer antigens
• has 19 antigens listed
• Inab phenotype- the Cromer null phenotype
• CROK (CROM19)- a novel Inab-like phenotype with a mutation that
affects the expression of the Cromer antigens
• Dr(a–) RBCs have weakened expression of all other high-prevalence Cromer
antigens due to a markedly reduced copy number of DAF.
• The Dr(a–) phenotype has been found only among Jews from Bakhara and in
Japanese
• antigens are resistant to treatment with ficin and papain but are destroyed by
α-chymotrypsin
• Cromer antigens are weakened with DTT treatment and are resistant to
glycine-acid EDTA
• Antibodies are usually IgG, but do not cause HDFN
• DAF is strongly expressed on placental tissue and will adsorb Cromer
antibodies
• Anti-Cra and anti-Tca have been implicated in HTRs
• Anti-IFC is the antibody made by individuals with the Cromer null Inab
phenotype
The Knops System
(022)
KN
• was named after Mrs. Knops, the first antibody maker
• was established when the antigens were shown to be located on
complement receptor 1 (CR1)
• The gene CR1 is located on chromosome 1q32.2
• There are nine antigens in the Knops system
• Knops antigens are weakly expressed on cord RBCs and weaken upon
storage of adult RBCs
• antigens are weakened by treatment with ficin and papain and are
destroyed by DTT; the antigens are resistant to glycine-acid EDTA
• The “Helgeson phenotype” represents the serologic null phenotype for
the Knops blood group these RBCs type Kn(a–b–), McC(a–), Sl(a–), and
Yk(a–) because of the low copy number of CR1
• Antibodies are usually IgG, reactive in the antiglobulin phase
of testing, but are clinically insignificant for both transfusion
and HDFN
- demonstrate variable reactions, are not neutralized by
pooled normal serum and are difficult to adsorb and elute.
Antibody reactivity is enhanced with longer incubation (e.g.,
1 hour, at 37°C)
The Indian System
(023)
IN
• was named because the first In(a+) individuals were from India in
1973
• There are now five antigens in the system
• Inb is the antithetical high-prevalence antigen
• antigens are located on CD44, an adhesion molecule
• The gene CD44 is located on chromosome 11 at position 11p13
• In(a–b–) phenotype has been found in only one individual who
presented with congenital dyserythropoietic anemia and whose
RBCs also typed Co(a–b–)
• CD44 is reduced on RBCs of dominant type Lu(a–b–) individuals
• Ina and Inb are weakly expressed on cord RBCs and are sensitive to
treatment with ficin, papain, and DTT but are resistant to glycine-
acid EDTA
• Antibodies are usually IgG and reactive in the antiglobulin phase of
testing and do not bind complement
• Positive DATs but no clinical HDFN have been reported for anti-Ina
and anti-Inb
• decreased cell survival with anti-Ina and an immediate HTR due to
anti-Inb have been reported
The Ok System
(024)
OK
• there are three high-prevalence antigens in the Ok system
• 1979: Anti-Oka was identified and was named after the antibody
maker, Mrs. Kobutso
• Two additional antigens, OKGV and OKVM
• OK antigens are carried on CD147, or basigin, a member of the
immunoglobulin superfamily
- Basigin is a receptor essential for invasion by Plasmodium
falciparum
• The OK gene BSG is on chromosome 19p13.3
• Oka is well developed on RBCs from newborns and is
resistant to treatment with ficin and papain, DTT, and
glycine-acid EDTA
• The original anti-Oka was IgG, reactive in the antiglobulin
phase of testing
• The antibody caused reduced survival of Cr-labeled Ok(a+)
RBCs injected into the original antibody maker, suggesting
clinical significance
• There have been no reports of HDFN caused by anti-Oka, but
this may be due to the rarity of the antibody
The Raph System
(025)
RAPH
• only antigen in the Raph system is MER2
• antigen name is derived from monoclonal, and Eleanor Roosevelt, the
laboratory where the antibody was produced
• the system was named Raph for the first patient to make the alloanti-
MER2
• MER2 is encoded by the gene CD151 located on chromosome 11p15.5
• Three examples of alloanti-MER2 were found in Jews originating from
India and living in Israel; two were related. All three had end-stage renal
disease.
• A fourth example of anti-MER2 was found in a healthy Turkish blood
donor
• MER2 is located on CD151, a tetraspanin, which appears to be essential
for the assembly of basement membranes in the kidney and skin
• MER2 expression decreases over time with increasing maturation of
erythroid cells
• The antigen is resistant to treatment with ficin and papain but is
sensitive to treatment with trypsin, a-chymotrypsin, pronase, and
AET
• Little data about the clinical significance of anti-MER2, but one
patient with the antibody showed signs of a transfusion reaction
after transfusion with 3 units
The John Milton Hagen System
(026)
JMH
• 1978: a large number of samples with antibody was characterized
and the antibody was named anti-JMH for the first antibody maker,
John Milton Hagen
• Numerous examples of anti-JMH have been seen, especially in
patients 50 years and older
• the JMH protein is the GPI-linked glycoprotein CD108
• The gene SEMA7A is located on chromosome 15q22.3-q23
• JMH is a high-prevalence antigen; Five other antigens were added
to the system, JMH2 through JMH6
• JMH1 represents the antigen recognized by antibodies made by
individuals lacking the JMH protein
• the patient’s JMH– status is acquired and can be transient
• JMH levels decline during the later years of life, sometimes to the
point of not being detected serologically
• JMH is weakly expressed on cord RBCs and is destroyed by treating
RBCs with ficin and papain and DTT; the antigen is resistant to
treatment with glycine-acid EDTA
• Anti-JMH is usually IgG (predominantly IgG4 in acquired JMH-
negative people)
• alloanti-JMH in individuals whose RBCs express variant forms of
CD108 may be clinically significant
The Gill System
(029)
GIL
• 1980: Anti-GIL was first identified
• GIL, the only one antigen, is genetically discrete from all other blood
group systems
• This antigen is found on the glycerol transporter aquaporin 3
(AQP3)
• The gene AQP3 is located on chromosome 9p13
• anti-GIL is enhanced with ficin and papain treatment of RBCs
• the antigen is resistant to DTT and glycine-acid EDTA treatment
• RBCs of two babies born to mothers with anti-GIL had a positive
DAT but no clinical HDFN
• One example of anti-GIL was found following a hemolytic
transfusion reaction
The Rh-Associated Glycoprotein
System
(030)
RHAG
• Rh-associated glycoprotein (RhAG): its presence in a complex with the
Rh proteins is essential for Rh antigen expression
• Absence of RhAG due to inactivating mutations in RHAG results in the
Rhnull phenotype
• Some missense mutations in RHAG result in the Rhmod phenotype
• RhAG is glycosylated and is encoded by the gene RHAG, located on
chromosome 6p12.3
• Four antigens have been assigned to the RHAG system:
- Duclos (high prevalence)
- Ola (very rare, low prevalence)
- DSLK (for Duclos-like)
- RHAG4
• Homozygosity for the allele encoding Ola is associated with the
Rhmod phenotype
• Absence of Duclos and DSLK is associated with U– Rhnull or U–
Rhmod phenotypes
• Anti-RHAG4 has caused a single case of severe HDFN, but
significance for transfusion is unknown
The FORS System
(031)
FORS
• 1987: the Forssman glycosphingolipid was first thought to be a
subgroup of A called Apae
• 2011: it was shown to be unrelated to the ABO system and renamed
FORS in honor of John Forssman, who first discovered the antigen
• only one antigen, FORS1
• The gene GBGT1 is located on chromosome 9 at position 9q34.13-
q34.3
• GBGTI gene  glycosyltransferase  N-acetylgalactosamine
(GalNAc) + P antigen = Forssman glycolipid
• Forssman glycolipid is not normally expressed on RBCs
- can serve as a receptor for pathogens such as Escherichia
coli
• FORS1 is enhanced with ficin and papain treatment, but
resistant to DTT and glycine-acid EDTA treatment
• The antibody is mostly IgM with optimal reactivity at room
temperature or 4°C, with an unknown clinical significance
The JR (032) System
(032)
JR
• 1970: The first anti-Jra, named for the antibody maker Rose
Jacobs, was described
• Jra is a high-prevalence antigen in most populations
• Jr(a–) phenotype is found more commonly in Japanese
• The gene ABCG2 is located on chromosome 4q22.1
- encoded protein, ABCG2, is a member of the adenosine
triphosphate (ATP) binding cassette transporters, involved in
multidrug resistance in tumor cells, presenting a problem in
chemotherapy
• The antigen is fully developed at birth
- is resistant to treatment with ficin and papain, DTT, and
glycineacid EDTA
• Anti-Jra is usually IgG
• Anti-Jra has caused severe HDFN in some cases, including
two fatal cases of HDFN
The LAN System
(033)
LAN
• 1961: Lan was first named after the first antigen-negative proband, Langereis,
who made anti-Lan
• was raised from the 901 series of high-incidence antigens to system status in
2012
• The Lan gene ABCB6 located on chromosome 2q36, encodes another of the
ATP-binding cassette transporters
• Cord RBCs have a stronger reaction with monoclonal anti-Lan than do adult
cells
• Reactivity with anti-Lan is resistant to ficin and papain treatment of RBCs; the
antigen is also DTT and EDTA/ glycine-acid resistant
• Anti-Lan are IgG formed due to exposure via transfusion or pregnancy,
reacting at the antiglobulin phase of testing; some antibodies known to bind
complement
• Anti-Lan is considered clinically significant
The Vel System
(034)
VEL
• 1952: Anti-Vel was first described and was named after the individual in
whom the first antibody was identified
• 2013: was raised to system status when absence of SMIM1, a single-pass
integral membrane protein, was shown to produce the Vel– phenotype
• The gene SMIM1 is located on chromosome 1 at position 1p36.32
• There is one antigen, Vel, and expression is weak on cord RBCs
• Reactivity with anti-Vel can be enhanced with enzyme-treated RBCs. The
antigen is also resistant to glycine-acid EDTA and DTT treatment
• Anti-Vel, most often IgG, is characterized by its ability to activate
complement and cause in vitro and in vivo hemolysis; it has caused
severe, immediate HTRs and caused one case of severe HDFN in which
the mother had previously been transfused
The CD59 System
(035)
CD59
• 2014: blood group number 035 was assigned to the CD59 system
based on a CD59-deficient child who formed an alloantibody with
CD59 specificity
• The gene CD59 is located on chromosome 11p13
• has only one antigen, CD59
- is a GPI-linked complement-regulatory glycoprotein also known as
the membrane inhibitor of lysis (MIRL)
• PNH patients are deficient in all GPI-linked proteins, including CD59
• Congenital CD59-deficient individuals show PNH-like symptoms,
including hemolysis and strokes, but also neuropathy
• One anti-CD59 was IgG and showed increased reactivity with
enzyme-treated RBCs and was nonreactive with DTT-treated RBCs
The Augustine System
(036)
AUG
• 1967: Anti-Ata was first described in the serum of a black
woman named Mrs. Augustine
• The system has two antigens:
AUG1
- the antigen defined by the antibody produced with the null
phenotype
AUG2 (Ata)
- is a high-prevalence antigen
 AUG3
- A new low-prevalence antigen (the corresponding antibody
caused severe HDFN)
• The AUG gene SLC29A1 located on chromosome 6p21.1
- encodes the equilibrative nucleoside transporter 2
(ENT2)

• The antigens are fully developed at birth

• Antigens are resistant to treatment with ficin and papain,
DTT, and glycine-acid EDTA
• Anti-Ata is usually IgG, reactive in the antiglobulin phase
of testing, and has caused severe HTRs and one reported
mild case of HDFN
ISBT Blood Group Collections
• Collections- are antigens that have a biochemical,
serologic, or genetic relationship but do not meet the
criteria for a system
• Most the antigens in the blood group collections are
either high or low prevalence and are assigned a 200
number
 Cost Collection 205
• formally referred to as Cost-Sterling, was established in 1988
• was named after the two patients (Copeland and Sterling) who made anti-Csa
• has been detected on three of four examples of the Helgeson phenotype
(Knops serologic null phenotype)
• currently consists of two antithetical antigens, Csa and Csb
• antigens are thought to be expressed on cord cells with a slightly weaker
expression of Csa
• antigens demonstrate a resistance to enzyme treatment and varying effects to
treatment with DTT.
• The Cost collection antibodies are IgG reacting at antiglobulin phase
 Ii Collection 207
• 1990: The Ii collection was first assigned and consisted of two antigens, I and I
• 2002: the I antigen was promoted to a blood group system 027
• i the sole antigen in the collection
- occurs on unbranched carbohydrate chains and is the precursor for the I
antigen
- strongly expressed on cord cells with only trace amounts on adult RBCs (i adult
phenotype is rare w/ anti-I present)
- resistant to both enzyme and chemical treatments
• Anti-i is usually IgM reacting at room temperature or 4°C
• Autoanti-i is a cold agglutinin associated with infectious mononucleosis and
some lymphoproliferative disorders
 Er Collection 208
• 1990: The Er collection was established as a collection with three antigens
(Era, Erb, and Er3)
• The Er antigens are expressed on cord cells
• Er antigens are resistant to enzyme and DTT treatment, but sensitive to
glycine acid/ EDTA treatment
• Anti-Er3 is produced by the presumed null phenotype Er(a−b−)
• antibodies are IgG reacting at the antiglobulin phase of testing and do not fix
complement
• Monocyte-monolayer assays (MMA) suggest anti-Era is not clinically
significant, whereas anti-Er3 had potential significance
 Globoside Collection 209
• currently consists of one highprevalence antigen, LKE
• relates to the P1PK (003) and Globoside (028) blood
group systems
 Collection 210 (Unnamed)
• consist of two antigens, Lec and Led, which are
glycosphingolipid adsorbed onto RBCs
• precursors to the Lewis antigens and demonstrate
increased expression in Le(a−b−) individuals
• neither antigens are produced by a Lewis gene transferase
• Anti-Lec agglutinates Le(a−b−) RBCs from nonsecretors
• Anti-Led agglutinates Le(a−b−) RBC from secretors
MN CHO Collection 213
• currently consists of six polymorphic antigens: Hu, M1, Tm,
Can, Sext, and Sj
• associated with the M or N antigen in the MNS (002) system
and are expressed on GPA with altered levels of sialic acid
(NeuNAc) or GlcNAc
• All the antigens are sensitive to ficin/papain, with some
demonstrating resistance to α-chymotrypsin
• All the antibodies to the MN CHO collection react optimally
at room temperature
• The Hu antigen (213001) was formerly called Hunter after the donor
of the RBCs used to immunize rabbits in 1934
- predominantly an altered GPAN with a strong link to the Sext
antigen
- Enzyme treatment shows sensitivity to ficin/papain and trypsin and
resistance to α-chymotrypsin
• The M1 antigen (213002) was reported in 1960 and is
predominantly an altered GPAM
- Enzyme treatment shows sensitivity to ficin/papain and trypsin and
resistance to α-chymotrypsin
- is expressed only on M+ RBCs
- anti-M1 are made by M– individuals
• 1965: the Sheerin antigen was renamed Tm (213003)
- predominantly an altered GPAN
- Enzyme treatment of the Tm antigen shows sensitivity to
ficin/papain and trypsin and resistance to α-chymotrypsin
• 1979: The Can antigen (213004) was reported and named
after the first antigen-positive proband, Canner
- predominantly an altered GPAM
- sensitive to ficin/ papain and trypsin and resistant to α-
chymotrypsin
• 1974: The Sext antigen (213005) was reported and named after the
individual who produced the antibody
- predominantly an altered GPAN with a strong link to the Hu antigen
- antigen is sensitive to ficin/papain, trypsin, and salidase
• 1968: The Sj antigen (213006) was reported when an anti-Tm serum
was shown to have an additional specificity
- was named after two employees at the New York Blood Center,
Stenbar and James, whose RBCs demonstrated strong agglutination
with the serum
- predominantly associated with GPAN and sensitive to ficin/papain
ISBT 700 Series
 ISBT 700 Series
• are low-prevalence antigens that represent those with a prevalence
of less than 1% of most random populations
• How the lowprevalence antigen/antibodies were detected:
1. an unknown maternal antibody causing HDFN
2. an unexplained incompatible crossmatch
3. unexplained positive reactivity with commercial human source
antisera containing an unknown low-prevalence antibody
• Seventeen antigens currently make up the 700 series of the
ISBT classification

• anti-HJK- A case of severe HDFN that required three

intrauterine transfusions was seen in a neonate due to its
presence
• anti-Kga and anti-REIT caused HDFN required exchange
transfusion
ISBT 901 Series
ISBT 901 Series
• Antigens in this series represent those with a prevalence of
more than 90% of most random populations
• finding compatible units negative for high-prevalent antigens
in the 901 series can be challenging
• Six antigens currently make up the 901 series of the ISBT
classification
• antibodies to the high-prevalence antigens a potential
transfusion risk
 Emm Antigen
• 1987: The Emm antigen was first reported and named after the
first identified antigen-negative proband
• The antigen is expressed on cord cells and is resistant to all enzyme
and chemical treatments
• Emm antigen is carried on a GPI-linked protein and (PNH) patients
are deficient in all GPI-linked proteins including the Emm antigen
• Anti-Emm has been identified to be both IgG and IgM reacting at
both 4°C and antiglobulin phase of testing with a higher occurrence
of IgG antibodies
 AnWj Antigen
• Anti-AnWj is an IgG antibody demonstrating reactivity at
antiglobulin phase of testing
• most anti-AnWj are autoantibodies due to a transient
suppression of the AnWj antigen
 AnWj Antigen
• 1982: alloantibody to an antigen called Anton was identified
• 1983 identification of an autoantibody to an antigen call Wj
• 1985, it was shown that they were the same antigen and renamed
AnWj
• AnWj antigen is not expressed on cord cells
- Is used by Haemophilus influenza as a receptor to enter RBCs
• RBCs of the dominant In(Lu) phenotype demonstrate a very weak
expression of the AnWj antigen
• antigen is resistant to ficin/papain, trypsin, and α-chymotrypsin, and
is variable with DTT
 Sd a Antigen
• is a high-prevalence carbohydrate antigen named for Sid, who was
the head of the maintenance department at the Lister Institute in
London
• The soluble form of Sda is Tamm-Horsfall glycoprotein found in urine
• antigen is not expressed on RBCs of newborns but is in their saliva,
urine, and meconium
- Markedly reduced during pregnancy
• Only 4% of people are Sd(a–)
• Strong examples of Sda are noted as Sd(a++)
 Sd a Antigen
• Anti-Sda- can naturally in individuals who are Sd(a–)
- usually an IgM agglutinin that is reactive at room temperature, but it can
be detected in the indirect antiglobulin test and does not react with cord
RBCs
• Reactivity is described as small, refractile (shiny) agglutinates in a sea of
free RBCs when viewed microscopically
• neutralization of the refractile agglutinates by urine is a technique used
to identify anti-Sda
• Sda antigen is resistant to treatment with ficin, papain, DTT, and
glycineacid EDTA
• Reactivity of the antibody is enhanced with enzyme-treated RBCs
PEL Antigen
• 1980: PEL antigen was first identified
• 1996: given the name PEL after the first negative
proband
• PEL antigen is expressed on cord cells and is resistant
to both enzyme and chemical treatment
• Anti-PEL are presumed to be IgG reacting at the
antiglobulin phase of testing
 ABTI Antigen
• 1996: the high-prevalence ABTI antigen was reported with the
detection of anti-ABTI in three multiparous women of an inbred
Israeli-Arab family
• The ABTI antigen is resistant to enzymes and chemical treatment
• Anti-ABTI are IgG reacting at the antiglobulin phase of testing
• Anti-ABTI has not demonstrated evidence of HDFN and clinical
significance of anti-ABTI for transfusion is unknown since there are
no clinical data available
 MAM Antigen
• 1993: MAM was first reported
• 1999: assigned to the 901 series
• Four MAM– probands have been reported and thought to
have formed anti-MAM through exposure during pregnancy
• MAM antigen is expressed on cord cells, lymphocytes,
granulocytes, monocytes, and probably platelets
• MAM antigen is resistant to both enzyme and chemical
treatment
• AntiMAM are IgG, reacting at the antiglobulin phase of
testing
 HLA Antigens on RBCs
• RBCs- generally do not have detectable levels of HLA antigens; thus HLA
antigens are not considered a blood group antigen
• The name “Bg” was given to HLA class I antigens that are detectable on mature
RBCs
- Bga: represents HLA-B7
- Bgb: represents HLA-B17
- Bgc: represents HLA-A28
• HLA antigens on RBCs are not destroyed by enzymes or DTT/AET treatment,
but chloroquine or EDTA/glycine-HCL can be used to remove the HLA antigen
from RBC
• HLA antibodies play a significant role in transfusion-associated acute lung
injury (TRALI)