MODULE VI (part 1, 2)

Minor Blood Groups

Ricky Martinez, RMT, BB (ASCP) SBB

 Intended Learning Outcome
For each of the blood group systems:
1. Describe the major antigens and antibodies of the minor blood group system
2. Enumerate the reasons for the clinically significance of the antibodies
3.. Describe the serologic characteristics and clinical significance of the
antibodies
4. Identify their null phenotypes
5. Identify any disease association of the blood group
• a. Lewis
• b. I / i
• c. M and N
• d. P
• e. Cold Auto antibodies
• f. Warm auto antibodies

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 Lewis Blood Group System
• In 1955, Sneath and Sneath3 observed that red cells lacking Lea and Leb will take up
these antigens from plasma containing them. Equally, red cells expressing either
Lea or Leb will give these up to plasma lacking them. In other words, the antigens
were found to be soluble.
• Between 1948 and 1951, Grubb4,5 and Brendemoen6 independently observed that
the saliva of Le(a+b-) individuals strongly inhibited anti-Lea, and that the saliva of
the majority of Le(a-b+) individuals also inhibited anti-Lea, but did so less strongly.
• In 1963, Mollison et al7 demonstrated that this phenomenon also occurred in vivo.
• From this it can be seen that:
• Lewis antigens are not intrinsic to red cells.
• They are located on type 1 glycosphingolipids that are adsorbed onto the red cells
from the plasma.
• Lewis, therefore, is not strictly speaking, a red cell blood group

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 Lewis System

• Not an integral part of the red cell membrane

• Manufactured by tissue cells and secreted into
body fluids then absorbed into red cells.
• Found in secretions such as saliva, milk and
plasma
 Lewis Antigens

• Le antigens (cell bound antigens) lipids

• Le substances (secretions) proteins
• Poorly developed at birth (Lea-Leb-)
• Le(a-b-)>>Le(a+b-)>> Le(a+b+)>>Le(a-b+)
• Can disappear during pregnancy
 Le Genes Inheritance

• Chromosome 19
• Formation depends on interaction between Lewis
(LeLe, Lele), Secretor (SeSe, Sese), and Hh genes in the
tissues to produce Lewis antigen in the secretions.
• Lewis antigens have similar structures to ABO antigens.
• 2 alleles – dominant – (LeLe, Lele) >>>Lea
• lele – recessive – Lewis negative
 Lewis Blood Group

• Lea and Leb - not antithetical antigens

• Inheritance of the Le gene results - development of
Lea regardless of the secretor status
• Inheritance of the Le, Se, and H genes homozygous
or heterozygous - result in Leb

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 Lewis Blood Group

• Lewis, saliva, plasma phenotype

• Saliva of individuals whose RBC contain Le(a+b-) -
contain Lea substance
• Saliva of individuals whose RBC contain
Le(a-b+) -contain Lea and Leb substance
Saliva of individuals whose RBC type as Le(a-b-)
does not contain Lea and Leb substance.

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 Non-Secretors

• Le(a+b-)
• Genes present
– Le gene only
– No Se gene (sese)
– May or not have the H gene
• All Le(a+b-) – non secretors of ABH substances
• Non-secretors – sese – 20%
 Secretors

• Le(a-b+)
• Genes present
– Le gene
– Se gene
– H gene
– Secretors – SeSe, Sese – 80%
 Formation of Lewis Antigen

• Type 1 – precursor substance

• Le gene – adds fucose to 2nd sugar >>>>Lea
• Secretor – H gene adds fucose to terminal
sugar >>>>>Leb
• Non-secretor – no H added to precursor
substance>>>>remains Lea
Type 1- Precursor
 H Structure

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 Lea

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 Leb

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 Lewis Antibodies
• Almost always produced by Lea-, Leb-
• Naturally occurring, IgM, non red cell stimulated
• Clinically insignificant
• Reacts at room temp
• Binds complement – in vitro hemolysis
• Enhanced by enzymes
• Le(a-b+) individuals do not make anti-Lea because
Lea antigen structure is contained within the Leb
antigen epitope.
• Anti-Lea - made by Le(a-b-) people.
• Neutralized by Lewis blood group substances
 Lewis Blood Group

GENES SECRETIONS RBC

Le, Se, A/B/H Lea, Leb, A,B,H Lea- Leb+ A,B,H

lele, Se, A/B/H A,B,H Lea- Leb- A, B, H

Le, sese, A/B/H Lea Le+ Leb- A,B, H

lele, sese, A/B/H none Lea- Leb- A, B, H

Le, Se, A/B/hh Lea Lea+ Leb- Oh

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 Summary

• Lea and Leb - not alleles

• Leb RBC arises - inheritance of Le, Se, H genes
• Lea+ Leb- non-secretors except bombay
• Bombay - cannot express Leb antigen
• Non-secretor (sese) can still secrete Lea into the
body fluids
• Glycoproteins - Lewis antigens in secretions
• Glycolipids - Lewis antigens in plasma
• RBCs absorb only glycolipids not glycoproteins onto
the membrane.
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 Testing for Secretor Status

• Agglutination Inhibition
• 1. patient’s saliva boiled and cleared
• 2. saliva mixed with anti-A, Anti-B, anti-H
• 3. If soluble A,B,H antigens present in saliva ----these
will react with antibodies in reagent -----will
neutralize it –no available antibodies to agglutinate
test cells.
• 4. If there are no soluble A,B,H antigens in saliva,
antibodies in the reagent will not be neutralized and
are free to react with test cells.
 Agglutination Inhibition

• Interpretation
• Agglutination – negative test
• No agglutination – positive test
 I/i Blood Group System
• I antigens:
• Located below the ABH antigens
• When ABH sugars are removed by some enzymes, I activity
increases.
• I antigens - branched Type 2 chains
• Bombay – greatest amount of I antigens
• i antigen – at birth and infancy
• i antigen - linear Type 2 chains
• 18 months of life – i antigen decreases I increases
• HEMPAS (hereditary erythroblastic multinuclearity with
positive acidified serum test) – associated with greater i
activity on red cells than cord cells.
 I Antibodies
• Benign anti-I – weak naturally occurring IgM
• Auto agglutinin reactive only at 4C
• Clinically insignificant
• Binds complement
• Anti-I demonstrates stronger reaction with red cells
having greater amounts of H - O and A2 red cells
• Pathologic anti-I – potent cold agglutinin reacts at
wide thermal range (0- 30C)
• Auto anti-I – mycoplasma pneumoniae
• Anti-i – IgM reacts at 4C
• Anti-i – infectious mononucleosis
 Introduction - Kell
• These erythrocyte antigens are the third most potent
immunogenic antigen after ABO and Rh system, and are
defined by an immune antibody, anti-K. It was first noticed in
the serum of Mrs. Kellacher. She reacted to the erythrocytes
of her newborn infant resulting in hemolytic reactions. Since
then 25 Kell antigens have been discovered. Anti-K antibody
causes severe hemolytic disease of the fetus and newborn
(HDFN) and hemolytic transfusion reactions (HTR)

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 Kell Blood Group System

• K (K1) - 9% frequency
• k (K2) - Cellano- 99-8% frequency
• Kpa (K3) - Penney - 2% frequency
• Kpb (K4) - Rautenberg - 99.9% frequency
• Jsa (K6) - Sutter - 20% (blacks), 0.1% whites
• Jsb (K7) - Matthews - 99% frequency
• Strongly immunogenic, ranks 2nd to D antigen

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 Kell Antigen Biochemistry

• Located on glycoprotein - integral part of the

red cell membrane
• K glycoprotein - linked to another protein - Kx
• Kx -defines the Kx blood group system
• Glycine acid-EDTA - sulfhydryl reagents that
creates red cells that lack Kell antigens.
 Kell Antigens

• Found on red cells

• Kx antigen – precursor for Kell antigens
• Well developed at birth
• Not destroyed by enzymes
• Destroyed by sulfhydryl reagents
– AET – 2-aminoethylisoronium bromide
– DTT – dithiothreitol
– 2 ME - 2 mercaptoethanol
– ZZAP – artificial Kell null
 Kell Antibodies

• IgG
• Clinically significant
• Reactive at AHG phase
• Cause HDFN and HTR
• Do not bind complement
• Shows no enhancement or depletion with
enzymes
• Common antibody – next to ABO and Rh
 Ko or Kell Null Phenotype

• Kell null is the result of - inheritance of 2

recessive genes - KoKo
• KoKo - lack all Kell antigens but express
another related antigen Kx antigen
• Antibodies produced by KoKo >>>> Anti-Ku
(anti-K5)
• Ku antigen - present on all red cells except Ko
cells

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 Kx Blood Group System

• Autosomal gene responsible for the Kell -

Chromosome 7
• Another gene XK1 -carries the Kx antigen
• Kx - genetically independent of the Kell
antigens
• Kx - posses phenotypic relationship to the Kell
blood group system
• Red cells from Ko people - elevated levels of
Kx antigens
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 Mcleod Phenotype

• XK1 is not inherited - there is no Kx antigen

• Absence of Kx antigens - means reduced
expression of the Kell blood group system -
known as Mcleod phenotype
• Mcleod phenotype - morphologic and
functional abnormalities
• rare - mostly in male population - due to X
chromosome borne gene.
• Chronic granulomatous disease (WBC
impaired) - associated with Mcleod phenotype
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 Introduction - Duffy
• Duffy-antigen was first isolated in a patient called Duffy who
had hemophilia. It is also known as Fy glycoprotein and is
present in the surface of RBCs. It is a nonspecific receptor for
several chemokines and acts as a receptor for human malarial
parasite, Plamodium vivax. Antigens Fya and Fyb on the Duffy
glycoprotein can result in four possible phenotypes, namely
Fy(a+b−), Fy(a+b+), Fy(a−b+), and Fy(a−b−). The antibodies are
IgG subtypes and can cause HTR

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 Duffy Blood Group System

• Duffy antigens – Fya and Fyb

• Well developed at birth
• Destroyed by enzymes
• Not found on granulocytes, lymphocytes,
monocytes, or platelets
• Fya- Fyb- >>>>> common among black people
• Receptors for P. vivax
 Genetics of the Duffy Blood Group

• Fy - located on chromosome 1
• Fy locus - synthenic with Rh locus
• Fya, Fyb >>>produce Fya and Fyb antigens
• Fyx -encodes weakened Fyb antigen
• Fy - encodes no identifiable Duffy antigen

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 Duffy Antibodies

• Anti-Fya, anti-Fyb
• IgG
• React at AHG phase
• Clinically significant
• Implicated with HDFN and DHTR
• Non reactive with enzymes
• Express dosage effect
• Anti-Fya more common than Anti-Fyb
 Introduction

• MNS antigen system, first described by Landsteiner

and Levine in 1927 is based on two genes:
Glycophorin A and Glycophorin B. The blood group is
under control of an autosomal locus on chromosome
4 and also under control of a pair of co-dominant
alleles LM and LN. Anti-M and anti-N antibodies are
usually IgM types and rarely, associated with
transfusion reactions

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 Duffy System and Malaria

• Fy(a-b-) red cells are not invaded by

– plasmodium knowlesi parasites
– plasmodium vivax
• Not plasmodium falcifarum

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 MNSs Antigens

• Found on chromosome #4
• Inherited as haplotype (M,N) & (S,s)
• Found on red cells, not in body fluids and secretions.
• MN – associated with glycophorin A
• Ss –associated with glycophorin B
• Well developed at birth
• Important markers in paternity testing
• MNSs antigens - genetic nature is caused by
• crossing over, gene recombination, substitution.
 MNSs Antibodies

• Anti-M and Anti-N

• IgM
• Naturally occurring
• Cold reactive
• Do not bind complement
• Destroyed by enzymes
• Shows dosage
 Anti-M and Anti-N

• Anti-M – reaction enhanced by acidification

• Shows dosage
• Stronger with M+N-, weaker with M+N+ cells

• Anti-N – seen in dialysis patients. Equipments

sterilized with formaldehyde.
 Anti-S, Anti-s

• Mostly IgG
• Reactive at 37 C and at the antiglobulin phase
• Bind complement
• Associated with HDFN and HTR
• S+, s+ have U antigen
• S-,s- are also U negative
 Introduction - Kidd

• Kidd antigen (known as Jk antigen) is a glycoprotein, present

on the membrane of RBCs and acts as a urea transporter in
RBCs and renal endothelial cells. Kidd antibodies are rare but
can cause severe transfusion reactions. These antigens are
defined by reactions to an antibody designated as anti-Jka,
discovered in the serum of Mrs. Kidd who delivered a baby
with HDFN. Jka was the first antigen to be discovered by Kidd
blood group system, subsequently, two other antigens Jkb and
Jk3 were found

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 Kidd Blood Group System

• Kidd Antigens
• Well developed at birth
• Causes HDFN
• In-vitro hemolysis
• Enhanced by enzyme treatment
 Kidd Antibodies
• Anti-Jka, anti-Jkb
• Show dosage
• Clinically significant
• IgG , reacts at AHG
• Binds complement
• Transient, causes delayed HTR
• Anti-Jk3- produced by Jka- Jkb- individuals.
• Jk(a-b-) null phenotype - Far East, Pacific Islanders
• Jk(a-b-) more resistant to lysis in the presence of 2M
urea than Jka+, or Jkb+ cells.
 P Blood Group System
Phenotypes Detectable antigens Possible antibodies

P1 P, P1 -

P2 P Anti-P1

p - Anti-PP1Pk

P1k P1,Pk Anti-P

P2k Pk Anti-P, Anti-P1

 P Antigens

• Consists of 3 antigens – P, P1, Pk

• P1 antigens are poorly developed at birth
• P1 and P2 are analogous to the A1 and A2
 P Antibodies

• Anti-P1
• IgM found in the sera of P2 individuals
• Weak reactive at cold temp
• Neutralized with hydatid cyst fluid
– (P1 substance)
 P Antibodies

• Anti-P - found in serum of P2 people

• Naturally occurring in all Pk individuals
• Enhanced by enzymes
• IgM cold reacting
• Auto-anti P – (Donath Landsteiner antibody)
IgG biphasic hemolysin – attaches to RBCs in
cold temp, lyses in warm temp)
• Associated with PCH – paroxysmal cold
hemoglobinuria.
 P Antibodies

• Anti-P, P1, Pk
– Igg and IgM
– Reacts at wide thermal range
– Binds complement
– Potent hemolysins
– Associated with spontaneous abortion
– In-vitro hemolysis
 Introduction - Lutheran

• Lutheran system comprised of four pairs of allelic

antigens representing single amino acid substitution
in the Lutheran glycoprotein at chromosome 19.
Antibodies against this blood group are rare and
generally not considered clinically significant

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 Lutheran Blood Group System

• Lutheran Antigens:
• Poorly developed at birth
• Show dosage effect
• Gene located on chromosome #19
• Closely linked with the secretor locus
• Resistant to enzymes (ficin, papain)
 Lu(a-b-)

• 3 different genetic backgrounds

• 1. dominant In-Lu types- expression of the
Lutheran is suppressed by a rare dominant
regulator gene In(Lu).
• 2. recessive lulu type – lacks all Lu antigens
• 3. recessive sex-linked inhibitor type-X-borne
inhibitor of Lutheran
 Lutheran Antibodies
• Anti-Lua
• naturally occurring
• React best at room temperature
• IgM or IgG
• Mixed field reaction
• Clinically insignificant

• Anti-Lub
• IgG immune antibodies
• React at AHG – 37C
• Bind complement
• Mixed field reactivity
• Associated with transfusion reactions.
• Mild cases of HDFN
 Other Minor Blood Groups

• Xg system- the only blood group produced under the

control of a gene located on the X chromosome.
• Bg system – related to HLA on RBCs
• Sd Antigen: found in saliva and urine of Sd +
• Anti-Sd:
– shiny, refractile, mixed field agglutination
– Neutralized by fresh human urine
 Other Minor Blood Groups
• Chido/Rodgers Blood Group:
• HTLA – high titer low avidity
• Clinically insignificant
• Diego Blood Group:
• Anti-Dia, anti-Dib
– IgG
– AHG phase
• Antigen: tool in anthropological studies of Mongolian
ancestry.
 Antibodies - HTLA

• Anti-Ch
• Anti-Rg
• Anti-Kn
• Anti-McC
• Anti-Yka
• Anti-Csa
• Anti-JMH
 References.
• Mourant AE. A ‘new’ human blood group antigen of frequent
occurrence. Nature 1946; 158: 237-238.
• Andresen PH. The blood group system L: a new blood group L2. A case of epistasy
within the blood groups. Acta Path Microbiol Scand 1948; 25: 728-731.
• Sneath JS, Sneath PHA. Transformation of the Lewis groups of human red
cells. Nature 1955; 176: 172.
• Grubb R. Correlation between Lewis blood groups and secretor status in man. Nature,
Lond 1948; 162: 933.
• Grubb R. Observations on the human blood group system Lewis. Acta Path Microbiol
Scand 1951; 28: 61-81.
• Brendemoen OJ. Studies of agglutination and inhibition in two Lewis antibodies. J Lab clin
Med 1949; 34: 538-542.
• Mollison PL, Polley MJ, Crome P. Temporary suppression of Lewis blood-group antibodies to
permit incompatible transfusion. Lancet 1963; 1: 909-912

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