IMHM311-LEC

WEEK 8: OTHER BLOOD GROUP SYSTEM-PART 1

MIDTERM I SECOND SEMESTER

LEWIS BLOOD GROUP SYSTEM Le (a- b-) phenotype:

• ISBT 007 • Results in point mutation of Le gene, rather than absence of
• Lewis gene (Le) codes for the production of the Le gene.
fucosyltransferase enzyme • These mutations give rise to a nonfunctional or partially
• This gene codes for a specific glycosyltransferase, a-4-L- active Lewis transferase (Lew ) causing the negative
fucosyltransferase which transfers L fucose to type 1 expression of the Lewis antigen on the RBCs.
chain (type 1 precursor) oligosaccharide on glycoprotein or • Presence of substances in secretions will depend on the
glycolipid structures secretor status of the individual (wether they inherit or not)
o Lewis blood group system is correlated with the alpha- o The Le (a- b-) nonsecretors express type 1 precursor,
4-L-fucosyltransferase which transfers the L-fucose as whereas the Le (a- b-) secretors express H type 1
immunodominant sugar to the type 1 precursor chain. substances plus ABH antigens associated with the
▪ Lewis blood group is in accordance with type 1 related ABO genes inherited.
precursor chain.
✓ Lewis blood group can be seen in secreted GENOTYPE SUBSTANCE IN THE RED CELL
substances. BODY FLUIDS PHENOTYPE
• Erythrocytes acquire the Lewis phenotype by adsorbing ABH, lele, sese NONE ABH Le (a- b-)
Lewis substances from the plasma, rather than membrane ABH, lele, SeSe or ABH ABH Le (a- b-)
SeSe
bound antigens.
ABH, LeLe or Lele, Lea ABH Le (a+ b-)
o Lewis gene: Se gene and H gene (located in the sese
chromosome 19) ABH, Lele or Lele, ABH, Lea, Leb ABH Le (a- b+)
SeSe or Sese
PRECURSOR MOLECULES Associated with body fluids involves in type 1 precursor chain
TYPE 1 PRECURSOR CHAIN
• beta 1-3 linkage DEVELOPMENT AND CHANGES OF LEWIS ANTIGENS
• the ABH antigens are formed from the same basic AFTER BIRTH
precursor materials and that is the Paragloboside • In plasma there are no detectable Lewis glycosphingolipids
• type 1 precursor chain attachment is between the terminal at birth.
of the D-galactose and N-acetylglucosamine o Therefore, cord blood and RBCs from newborn infants
• functions phenotype as Le (a- b-)
o beta 1-3 linkage produces ABH antigens depending to ▪ Undetectable at first
an individual if he/she is secretor or non-secretor • Infants that inherit LE and Se genes:
o secreted substances is associated with the type 1 Le (a − b −) → 𝐿𝑒 (𝑎 + 𝑏 −) → Le (a − b+)
10 𝑑𝑎𝑦𝑠 6−7 𝑦𝑒𝑎𝑟𝑠
precursor chain • Infants that inherit the Le and se genes:
▪ if an individual inherits the Se gene it codes for Le (a − b −) → 𝐿𝑒 (𝑎 + 𝑏 −)
the production of a-2-L-fucosyltransferase 10 𝑑𝑎𝑦𝑠

TYPE 2 PRECURSOR CHAIN DURING PREGNANCY

• A decrease in expression of Lewis antigens has been
• beta 1-4 linkage
demonstrated on RBCs from many pregnant women,
• located between the terminal D-galactose and N- resulting in Le (a- b-) phenotypes during gestation.
acetylglucosamine
• ABH antigens within the type 2 is primarily intended for the CHARACTERISTICS OF LEWIS ANTIGEN
red blood cells • Poorly developed at birth
o Construction of oligosaccharides chain within the type • Reversibly adsorbed onto red cells from plasma
2 precursor substance • Not found on cord blood or newborn red cells Le (a-b-)
o Resemblance of ABH antigen within the RBCs • Lewis glycolipids detectable in plasma after approximately 10
• Formation of ABH antigen are from the type 2 precursor days of life
chain • Transformation of Lewis phenotype after birth seen in
individuals who inherit Le and Se genes: Le (a- b-) to Le (a+ b-)
to Le (a+ b+) to Le (a- b+) (the true phenotype)
• Decrease in expression demonstrated in red cells from many
pregnant women, resulting in Le(a-b-) phenotype during
gestation
• Do not show dosage in serologic reactions

NOTE
• Exhibit the dosage within the serologic reaction: Rh (except
FIGURE 8–1 Structure of type-1 and type-2 chains. Gal = D-galactose; D antigen), Kidd, Duffy, MNSs
GlcNAc = N-acetyl-D-glucosamine; R = other biochemical residues.
Lewis Antibodies
• The inheritance of the Le gene acts in competition with • Naturally occurring
ABO genes, adding L-fucose to the GlcNAc sugar of the • IgM in nature
common precursor structure manufactured by tissue cells. • Complement activation (+) → can cause in vivo and in vitro
o The structure formed is known as Lea soluble hemolysis.
antigen
• Reactivity is enhanced when treated with proteolytic
o GlcNAc sugar → used for the specificity of the cell
enzymes.
• It is then secreted and adsorbed onto the RBCs,
lymphocytes, and platelet membranes from plasma. THE EFFECT OF PROTEOLYTIC ENZYMES ON SELECT
• Lewis antigen is also found on other tissues, such as the ANTIGEN-ANTIBODY REACTIONS
pancreas, stomach, intestine, skeletal muscle, renal cortex, Enhanced Inactivated
and adrenal glands. • Rh • Duffy
• Kidd • MNS
LEWIS PHENOTYPES • Lewis • Xga
Le (a+ b-) phenotype • P1
• Lea substance is secreted regardless of the secretor status. • I
• All Le (a+ b-) individuals are nonsecretors of ABH • ABO
substances. Kell (not affected by proteolytic enzyme)

Le (a- b+) phenotype: Anti-P k

• The genetically independent Sese, ABO, Hh , and Lewis
genes are intimately associated in the formation of the Leb
antigen.
• It still produces Lea substance
• The phenotype Le (a b+) is the result of the genetic
interaction of Lele and Sese genes.
• Isolated from some examples of anti-PP1Pk by selective
adsorption with P1 cells
• Has been reported in the serum of P1 individuals with
biliary cirrhosis and AIHA (autoimmune hemolytic
anemia)
• Anti-Pk activity can be inhibited with hydatid cyst fluid

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 WEEK 8: OTHER BLOOD GROUP SYSTEM
CHARACTERISTICS OF LEWIS ANTIBODIES
• Usually naturally occurring
• Predominantly IgM
• May cause in vivo hemolysis of red cells
• Sometimes reacts at 37°C and Coombs phase more weakly
than at room temperature
• Enhanced by enzymes
• Readily neutralized by Lewis blood group substances
• Rarely causes in vitro hemolysis; however, in vivo
posttransfusion hemolysis reported in cases in which Lewis Ab
strongly reacts in Coombs phase
ANTI-LEA
• Most commonly encountered antibody of the Lewis system.
• Produced by Le (a- b-) phenotypes.
• MOSTLY IgM. Some may have IgG form but does not bind
to the RBCs as efficiently as IgM anti-Lea form.
o It activates at room temperature or even colder
• The Lea antibody is frequently detected with saline-
suspended cells at room temperature.
• Sometimes react at 37C and Coombs (AHG) phase.
• Easily neutralized with plasma or saliva that contains Lea
substance.
• Phenotype Le (a- b+): do not make anti-Lea .
o It has a small amounts of unconverted Lea present in
the plasma and saliva.
• Le (a- b+) do not make anti-Lea production (no anti-lea)
ANTI-LEB
• Not as common and not as strong as anti-Lea
• It is also an IgM
• Usually produced by Le (a- b-)
o Occasionally produced by Le (a+ b-)
o The two are associated within each other
• Neutralized by plasma or saliva containing Leb substance.
LEWIS ANTIGEN ON THE RBCS AND POSSIBLE ANTIBODIES
PRODUCED IN THE PLASMA
Red Cell Phenotype Possible Antibodies Produced
Le (a- b-) Anti-Leb
Le (a- b+) - En (a-) antigen
Le (a- b-) Anti-Lea, Anti-Leb
Lex - o In 1969 Darnborough and coworkers and Furuhjelm
CLINICAL SIGNIFICANCE OF LEWIS ANTIBODIES
• Some cases of HTR caused by anti-Lea have been reported
• Cases of in vivo RBC destruction due to anti-Leb also have
been reported
• Generally, Lewis antibodies are considered insignificant in blood
transfusion practices
o It can be neutralized by the Lewis substances present in
the plasma and can thereby be decreased in quantity
o Lewis antigens dissociates from the RBCs as readily as
they bind to the RBCs
o Lewis antibodies are generally IgM and therefore cannot
cross the placenta and cause HDN
▪ Lewis antigens are not fully developed at birth
FACTORS CONTRIBUTING TO CLINICAL INSIGNIFICANCE OF
LEWIS ANTIBODIES
• Neutralization of Lewis antibodies by Lewis substances present
in the plasma
• Loss of red cell Lewis antigen(s) into the plasma
• Lack of reactivity at 37C and antihuman globulin phase
• Generally, IgM in nature and incapable of crossing placenta
• Lewis antigens poorly developed in newborn infants
MNSs Blood Group System
• ISBT 002
• Discovered by Landsteiner and Levine in 1927: M and N
antigens
o Immunizing rabbits with human RBCs.
o Data from family studies suggested that M and N were
antithetical antigens.
▪ Antithetical antigens - describes a pair of
antigens that are coded by different alleles of a
single gene.
• In 1947: Walsh and Montgomery discovered S antigen that
appeared genetically linked to M and N.
o Antithetical partner of S is the s antigen. (Discovered
in 1951).
• 1953: Wiener discovered U antigen.
M and N Antigen
• Found on a well characterized glycoprotein known as GPA
(glycoprotein A)
o major RBC sialic acid rich glycoprotein
• Well developed at birth
• Found on RBCs BUT NOT in platelets, lymphocytes,
monocytes and granulocytes
Pete Laurenz Gonzales
• Easily destroyed or removed inactivated by proteolytic
enzymes
o The proteolytic enzymes enhanced as Rh, Kidd,
Lewis, P1, I and ABO.
o The inactivated are Duffy, MNSs, Xga.
o Not affected by the proteolytic enzyme (Kell)
• Because M and N are located at the outer end of GPA
o M and N antigens is associated with amino acid
residue.
▪ M antigen it has a serine protein (1st position)
and glycine (5th position)
▪ N antigen it has leucine (1st) and glutamic acid
(5th)
• Antigens M and N differ on the 1st and 5th amino acids
• Shows dosage in serologic reactions
o Exhibit the dosage within the serologic reaction: Rh
(except D antigen), Kidd, Duffy, MNSs
S and s antigens
• Located on smaller glycoprotein called GPB (glycophorin B)
• S and s are differentiated by the amino acids at position 29
on GPB.
• S has methionine whereas s has threonine
• Well developed at birth
• Destroyed or removed/inactivated by proteolytic enzymes.
o Except for Trypsin → does not destroy S and s
antigens.
▪ If it is associated with Trypsin → Does not
destroy with the proteolytic enzyme
• Found on RBCs BUT NOT in platelets, lymphocytes,
monocytes and granulocytes.
OTHER MNSs ANTIGENS
U antigen:
• Located on GPB
• found on RBCs of all individuals except about 1 % of
American blacks (and 1-35 % of African blacks)
• U antigen negative cells are also S-s- (Ss Null)
• En code for envelope
and colleagues described an antibody to the same
high prevalence antigen, called Ena (for envelope),
which reacted with all RBCs except those of the
propositus.
• High Frequency antigen
• Failure to produce Glycophorin A results in Ena negative
red cells
o Both En (a-) individuals appeared to be M-N- with
reduced NeuNAc on their RBCs.
o The RBCs of the two individuals were mutually
compatible.
MNSs ANTIBODIES
Anti M:
• Naturally occurring
• IgM or IgG
• Do not bind complement: regardless of their Ig class.
• Do not react with enzyme treated RBCs.
• Demonstrate dosage
o Reacts better with M+ N- than M+ N+ RBCs.
• Enhanced reaction at pH 6.5.
• Some react only with red cells exposed to glucose solution
• Not clinically significant unless it reacted at 37C or AHG
phase.
o Rarely causes HDN.
Anti-N
• Naturally occurring
• IgM or IgG
• Do not bind complement: regardless of their Ig class.
• Do not react with enzyme treated RBCs.
• Demonstrate dosage
o Reacts better with M- N+ than M+ N+ RBCs.
• Rarely reacts with red cells exposed to glucose solution
• Not clinically significant unless it reacted at 37C or AHG
phase.
o Rarely causes HDN.
• Anti-N reagent used in laboratory: Vicia graminea (lectin
source)
Anti-Nf
• seen in renal patients, regardless of their MN type, who are
dialyzed on equipment sterilized with formaldehyde.
o Seen in Equipment used in dialysis
• reacts with any N+ or N- RBC treated with formaldehyde.
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• The antibody titer decreases when dialysis treatment and • Associated with Fascioliasis (liver fluke), Clonorchis
exposure to formaldehyde stop. sinensis (Chinese or oriental liver fluke) and Opisthorchis
• clinically insignificant in transfusion. viverrine infection.
• HDN is not associated with anti-P1
Anti-S and Anti-s
• IgG in nature Anti-PP1Pk
• They may bind complement • Originally called Anti-Tja
• they have been implicated in severe hemolytic transfusion o Originally called anti-Tja, anti-PP1 Pk was first
reactions with hemoglobinuria. described in the serum of Mrs. Jay, with cancer tumor,
• Also caused HDN a Pnull individual with adenocarcinoma of the
• Units selected for transfusion must be antigen negative and stomach.
crossmatch compatible. o Her tumor cells carried P system antigens, and the
• it can be difficult providing blood for a patient with anti-s. antibody was credited as having cytotoxic properties
that may have helped prevent metastatic growth post-
Anti-U surgery (the T in the Tja refers to tumor).
• is typically IgG and has been reported to cause hemolytic • Predominantly IgM, sometimes IgG
transfusion reactions and HDN or Erythroblastosis fetalis. • Reacts over a wide thermal range
• Efficiently bind complement, which makes them potent
Anti-Sera Reagents hemolysins.
• Anti-M • Anti-PP1Pk has the potential to cause severe hemolytic
o Human source transfusion reactions and HDN.
o Rabbit source • ASSOCIATED WITH SPONTANEOUS ABORTIONS IN
o Monoclonal source EARLY PREGNANCY
• Anti-N o Complete abortion
o Monoclonal source • Anti PP1Pk → reacts with all RBCs except to the auto
o Vicia graminea (Lectin source) control and those of the p phenotype.

FREQUENCY OF MNS PHENOTYPES Alloanti-P

Phenotype Whites (%) Blacks (%) • Naturally occurring
M+ N- 28 26 • Seen in sera of all Pk individuals.
M+ N+ 50 44 • Reactivity is the same as Anti PP1Pk
M- N+ 22 30 o Anti-PP1Pk→ reacts with all RBCs except to the auto
S+ s- 11 3
control and those of the p phenotype.
S+ s+ 44 28
S- s+ 45 69
• Rarely seen in blood bank (but very significant in
S- s- U- 0 <1 transfusion)
• Has been associated with habitual early abortion.
P Blood Group System
• P Blood Group Antigens ISBT 003 Autoanti-P
• P1, P and Pk antigens • Cold reactive IgG autoantibody
• Discovered by Landsteiner and Levine (1927). • Associated with PCH (paroxysmal cold hemoglobinuria)
o Injected human RBCs to rabbits and produced an • The IgG autoantibody in PCH is described as a biphasic
antibody. hemolysin.
o They called it as Anti-P o In vitro, the antibody binds to RBCs in the cold, and
• 1951, Levine et al. via complement activation, the coated RBCs lyse as
o Describe an antibody known as anti Tja ( now known they are warmed to 37C.
as Anti-PP1Pk o The autoantibody typically does not react in routine
▪ Made by Pnull individual. test systems but is demonstrable only by the Donath
Landsteiner test.
PHENOTYPE DETECTABLE POSSIBLE ANTIGENS
ANTIGENS DONATH-LANDSTEINER TEST
P1 P & P1 NONE 1. Collection of fresh blood from the patient
P2 P Anti-P1 2. Prepare three sets of test tubes
Pnull NONE Anti-P and anti-P1, anti- a. A1, B1, and C1 → add 10 drops of patients serum,
Pk 1 volume of 50% suspension of washed Group O
P1K PK, P1 Anti-P cells that expressed P antigen
P2K PK Anti-P and Anti-P1 b. A2, B2, and C2 → add 10 drops of patients serum,
10 drops of normal serum and 1 volume of 50%
P BLOOD GROUP: PHENOTYPES, ANTIGENS AND ANTIBODIES suspension of washed Group O cells that
Phenotype Antigens Possible Frequency
expressed P antigen
Present Antibodies Whites Blacks
c. A3, B3 and C3→ add 10 drops of normal serum,
P1 P1, P1 (Pk) None 79% 94%
P2 P1 (Pk) Anti-P 21% 6%
one volume of 50% suspension of washed Group O
p None Anti-PP1Pk Rare Rare cells that expressed P antigen
P1k P1, Pk Anti-P Very rare Very rare 3. Incubate
P2k Pk Anti-P, Very rare Very rare a. Incubation “A” tubes: Ice bath for 30 minutes and
Anti-P1 1 hour at 37C
b. Incubation “B” tubes: Ice bath for 90 minutes
P1 Antigen c. Incubation “C” tubes: 37C for 90 minutes
• Changes during fetal development. 4. Centrifuged and observed the supernatant for hemolysis
• Found on fetal red cells as early as 12 weeks, but it
weakens with gestational age Tube 1 Tube 2 Patient Tube 3
• Not well developed at birth Patient Serum and Normal
o Only Well-developed at birth are: MNSs, Kell, Kidd, Serum (tubes Normal Serum Serum (tubes
A1, B1, C1) (tubes A2, B2, C2) A3, B3, C3)
Duffy
Ice bath
• P1 antigen deteriorates rapidly on storage and 37C
• P1 like antigen has been found plasma and droppings of (all A
+ + 0
pigeons and turtle doves, as well as in the egg white of tubes)
turtledoves Ice bath
• P1 in hydatid cyst fluid, extracts of Lumbricoides terrestris only (all B 0 0 0
and Ascaris suum tubes)
o Parasite → Echinococcus granulosus (hydatid cyst) 37C only
(all C 0 0 0
tubes)
Anti-P1 + = with hemolysis
• Common, naturally occurring IgM antibody in the sera of P1 0 = without hemolysis
individuals Tubes with normal serum are used as control
• COLD REACTIVE (IgM room temperature or colder)
• Strong anti P1 was observed in individuals infected with
Hydatid dse
• Not well developed at birth

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DIAGRAMS

FIGURE 8–2 Formation of Lea substance.

FIGURE 8–3 Action of Le gene transferase enzyme.

Lea = gene codes for L-fucosyltransferase, which adds L-fucose (FUC) to the carbon number 4 of N-acetylglucosamine of type-1
precursor structure.
Type-1 H substance = Se gene codes for L-fucosyl transferase, which adds L-fucose to the carbon number 2 of terminal galactose.
Leb = in the presence of Le, Se, a second fucose is added to the carbon number 4 of the subterminal N-acetylglucosamine of type-1 H
substance.
Gal = D-galactose; GlcNAc = N-acetyl-D-glucosamine; Fuc = L-fucose; R = other biochemical residues.

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FIGURE 8–6 Formation of Lewis antigens in secretors and nonsecretors.

Gal = D-galactose; GlcNAc = N-acetyl-D-glucosamine; FUC = L-fucose; R = other biochemical residues

PROCESS:
• β-Gal(1→3) β-GlcNAc(1→3)LC (type 1 precursor molecules Beta-1,3 linkage)
o Le gene (a-4-L-fucosyltransferase) → Lewis gene will add fucose on the fourth carbon of the GlcNAc
o Se gene (a-2-L-fucosyltransferase) → While Se gene will add Fucose on the second carbon of b-Galactose
▪ A gene (a3-N-acetylgalactosaminyltransferase) → Will add GalNAc on Carbon 3 of b-galactose of type 1 precursor
molecule
L-Fuc → Still, it has Fucose molecule, since Se gene is present (SECRETOR)
✓ Le gene (seen in B type 1) → Presence of Le gene will add another fucose on the fourth carbon of GlcNAc
▪ B gene (a3-D-galactosaminyltransferase) → Will add Galactose on Carbon 3 of β-galactose of type 1 precursor
molecule
✓ Le gene (seen in A type 1) → Presence of Le gene will add another fucose on the fourth carbon of GlcNAc

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