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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_2617_Библиотеки_им_академика_М_И_Перельмана
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The use of the antiglobulin test, rst described by
Moreschi in 1908 and rediscovered in 1945 by Robin
Coombs, Rob Race, and Arthur Mourant, allowed the identication of many other blood group antigens in the decades
that followed. Currently, several hundred antigens are known
to be present on the surface of erythrocyte, classied in 33
blood group systems, 7 blood group collections, 700 lowincidence antigens, 901 high-incidence antigens.
Blood Group Antigens
The antigenic determinants of the erythrocyte blood group
systems are inherited, usually, following the dictates of formal genetics. During cell division (Mitosis), chromatin loses
its homogeneous structure and the chromosomes in which
the units of genetic information are present, called genes.
The genes are distributed with a specic order along the
chromosome, in a precise physical location called the locus.
Alternative forms of genes, each of which can occupy a single locus on one of the homologous chromosomes, are
termed alleles.
The ISBT terminology distinguishes between the alleles
for blood group antigens and the antigens that they encode.
For example, the major antigens of the ABO system are A, B,
and O, yet the alleles are A, B, and O. In the Kell system, two
alleles, K and k, determine the K and k antigens, respectively.
In other cases, the name of the gene alleles is very different
from that of the antigens they determine. For example, in the
MNSs blood group system, the genes are named GYPA and
GYPB. Almost all the gene alleles coding for the antigens of
the erythrocyte blood group systems are in one of the 22
pairs of autosomes, only one allele coding for the Xg and Xk
antigens has been located on the X chromosome.
Individuals who have identical alleles at a given locus on
both chromosomes (i.e., K/K) are homozygous. In heterozy-
gous condition, alleles present at the locus on each chromosome are nonidentical (i.e., K/k). Individuals who are
homozygous for an allele, in some blood group systems, may
have more antigen expressed on their red cells than persons
who are heterozygous. For example, red cells from a person
whose phenotype is Jk(a+b–) have a “double dose” of the
Jka allele and, as a result, express more Jka antigen on the
red cell surface than an individual whose phenotype is
Jk(a+b+). The difference in amount of antigen expressed on
the red cell membrane between a homozygous and a heterozygous phenotype sometime can be detected serologically
(dosage effect).
Usually blood group antigens are expressed as codominant characters. Therefore, heterozygotes express the product of both alleles. For example, if an individual’s
erythrocytes are typed as K + and k +, a K/k genotype can
be inferred.
In ABO system, the situation is more complicated. In fact,
the genes of the ABO system do not code for membrane proteins, but control the production of enzymes: glycosyltransferases. These enzymes add sugars to a preexisting
oligo-carbohydrate chain, modifying it and giving it the antigenic specicity A or B or leaving it unchanged (O). The
antigens of the ABO system are therefore oligosaccharide
chains. The determinants of the other erythrocyte blood
group systems are usually the glycoproteins present on the
erythrocyte membrane of which they are often integral constituents (intrinsic proteins).
Effectiveness of an antigen in evoking the immune
response is due to numerous characteristics, the total effect
of which is dened as immunogenicity. Some of these characteristics are the exposure of the antigen and the number of
determinants present on the erythrocyte membrane, maturation of the antigen during fetal life, its spread in the population, and more. Some relevant data concerning blood groups
antigens are reported in Table16.1.
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Table 16.1
ISBT
number
001 ABO ABO ABO 9 4 –
002 MNS MNSs GYPA, GYPB 4 46 CD235
003 P P P1 22 1 CD77
004 Rh Rh RHD, RHCE 1 52 CD240
005 Lutheran Lu LU 19 20 CD239
006 Kell K KELL 7 14 CD238
007 Lewis Le FUT3 (Le) 19 6 –
008 Duffy Fy FY 1 5 CD234
009 Kidd Jk JK 18 5 –
010 Diego Di AEI (SCL4AEI) 19 22 CD233
011 Cartwright Yt ACHE 7 2 –
012 Xg Xg XG X 2 CD99
013 Scianna Sc SC 1 7 –
014 Dombrock Do DO 12 8 CD297
015 Colton Co AQP1 7 4 –
016 Landsteiner-Wiener LW LW 19 3 CD242
017 Chido-Rodgers Ch/Rg C4A, C4B 6 9 –
017
019 Kx Kx XK X 1 –
020 Gerbich Ge GYPC 2 11 CD236
021 Cromer Cromer DAF 1 17 CD55
022 Knops Kn CR1 1 4 CD35
023 Indian In CD44 11 4 CD44
024 OK OK CD147 19 3 CD147
025 RAPH MER2 MER2 11 1 CD151
026 JMH JMH SEMA7A 15 6 CD118
027 I I CGNT2 6 2 –
028 GLOB P B3GALT3 3 1 –
029 Gil GIL AQP3 9 1 –
030 Rh associated glyccoprotein RHAG RHAC 6 4 CD241
031 Forsman FORS CBCT1 9 1 –
032 JR JR IJ, ABCC2 4 1 –
033 LAN LAN LAN, ABCB6 2 1 CdW338
034 Se Secretore FUT2 19 2
The rst column shows the numerical nomenclature of the International Society of Blood Transfusion (ISBT), in the second the full name of the
erythrocyte blood group system using the ISBT nomenclature, in the third column is reported the common name of the blood group systems. The
fourth column shows the name of the genes and the fth the location at chromosomal level. In the sixth the number of antigens and in the last
column the clone denomination (CD)
Blood group systems with gene denomination and chromosome location
ISBT
denomination
Hh H FUT1 19 1 CD173
Conventional
denomination
Genes
denomination
Localization
chromosome
Number of
antigens
Clone
denomination
Antibody Versus Blood Groups Antigens
Immunoglobulins (Ig) of prevailing immunohematological
interest are IgM and IgG. IgMs are pentameric antibodies
produced during the primary phase of the immune response,
and which in immuno-hematology are classically identied
with the “natural” antibodies of the ABO system. They are
complete antibodies, being able to generate agglutination in
physiological solution, and are extremely effective in activating complement according to the classical way. They are
unable to cross the placenta, while they can react well at 4°C
and 22°C.
IgGs are monomeric antibodies. In immunohematology,
the IgG1 and IgG3 subclasses are particularly relevant, to
which most of the allo-antibodies directed toward antigens
belonging to the erythrocyte blood group systems belong.
IgGs are produced during the secondary phase of the immune
response, and in immunohematology, they are identied
with immune antibodies. They are incomplete antibodies,
not being able to generate agglutination in saline solution.
Their presence is highlighted either by modifying the reaction conditions (low-ionic-strength solutions, polybrene,
albumin) or with the antiglobulin test. IgG can cross the placenta. Their thermal optimum is from 22 to 37°C.
In Transfusion Medicine, an antibody is dened clinically
signicant if associated, with a certain frequency, with
hemolytic posttransfusion reaction (PTH) and/or foetalneonatal hemolytic disease (HDFN).
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Antibody Antigen Reaction inErythrocyte
Immunohematology
Classical erythrocyte immunohematology is based on the
detection of reaction between the antigens present on the surface of the red blood cells and the antibodies present in the
antisera (or in patient serum). The antigen–antibody reaction
can produce a variety of observable results. In blood group
serology, the most observed reactions are agglutination,
hemolysis, and precipitation.
Agglutination is the most widely used antigen–antibody
reaction in erythrocyte immunohematology and consists of
the aggregation, mediated by antibodies, of red blood cells
that have the relative antigen on their surface. Agglutination
of red blood cells occurs because the antibody molecules
bind to the antigenic determinants of different but adjacent
red blood cells, binding them together to form a visible
aggregate.
Hemolysis consists of the rupture of red blood cells,
resulting in the release of intracellular hemoglobin. In vitro
hemolysis is a two-stage reaction: In the rst stage, there is
the binding of antibody to the blood group antigens; and in
the second stage, there is activation of complement system
leading to RBC destruction. Hemolysis is considered a positive result, because it demonstrates the antigen–antibody
reaction with activation of the complementary cascade.
Precipitation consists of the formation of an insoluble
complex, usually visible, which originates from the reaction
of a soluble antibody with a soluble antigen. These insoluble
antigen–antibody complexes are detectable in tube tests as
an annular or button-shaped sediment or in agar tests as a
precipitation line (Fig.16.1).
Fig. 16.1 Agglutination, hemolysis and precipitation
Erythrocyte Blood Group Antigenic Systems
ABO, H, andLewis Blood Groups
andStructurally Related Antigens
ABO blood group antigens, as well as those of the Hh, Lewis,
and Ii systems, consist of structurally related polysaccharide
molecules. The antigens are formed by specic glycosyltransferases, which add, in sequence, specic sugars on oligosaccharide chains that derive from a common precursor
substance. The interactions of the products of the ABO, Hh,
Sese,Se/seand Lele genes affect the expression of the ABO,
H, and Lewis antigens, as well as the presence of substance
A and B in body secretions.
The ABO system was discovered when Karl Landsteiner
recorded the agglutination of human red cells by the sera of
other individuals in 1901 and detailed the three patterns of
reactivity called groups A, B, and O.He found that serum
from group A individuals agglutinated the red cells from
group B individuals, and conversely, the serum from group B
individuals agglutinated group A. Red cells that were not
agglutinated by the serum of either the group A or group B
individuals were later called group O; the serum from group
O individuals agglutinated the red cells from both group A
and group B individuals. A year later a fourth group, named
AB was described. Serum of group AB individuals did not
agglutinate group A, B, and O red cells; conversely, the red
cells from group AB individuals were agglutinate from group
A, B, and O serum. From these observations derived two
fundamental concepts in immunohematology: antibodies to
A and B antigens are present when the corresponding antigen is missing and that these antibodies are almost always
present in people who have had no exposure to human red
cells (natural antibodies) (Fig.16.2).
The loci of the genes H and Se (Secretor), identied,
respectively, as FUT1 and FUT2, are located, in close concatenation, on chromosome 19. Each locus can be occupied
by two alleles, one of which, respectively, called h and se, is
an amorphous gene. The active alleles H and Se encode two
glycosyltransfers, which, by acting on a precursor, transform
it into H antigen. The H gene encodes a transferase that acts
at the cellular level to form the H antigen on red blood cells.
The transferase encoded by the Se gene also produces the H
antigen, but in secretions, such as saliva. The amorphous h
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Fig. 16.2 The Karl Landsteiner experiment. Serum from group A individuals agglutinated the red cells from group B individuals, and, conversely, the serum from group B individuals agglutinated group A.Red
cells that were not agglutinated by the serum of either the group A or
group B individuals were later called group O; the serum from group O
genes and if they behave like recessive genes, the h gene is
extremely rare.
The ABO locus is located on chromosome 9, and there
are three common alleles: alleles A and B encode transferases, which produce antigens A and B, respectively; the O
allele does not code for a functional enzyme. The red blood
cells of individuals of group O are therefore devoid of antigens A and B, but have a high quantity of antigen H.The
antigens of the ABO system are oligosaccharide chains,
which can be joined to other macromolecules to form glycoproteins, glisngolipids, glycolipids. The methods of conjugation affect the distribution of antigens. For example, ABO
antigens present on the surface of erythrocytes are usually
glycoproteins and glycosphingoplipids, in saliva they are
present as glycoproteins, in milk they are present as free
oligosaccharide.
Glycosyltransferases encoded by alleles A, B, H, and
Se add a specific sugar to a preexisting oligosaccharide
chain. This additional carbohydrate determines the antigenic specificity, which is, in fact, lost when it is removed
(referred to as immunodominant). These reactions can
only occur sequentially; therefore, the H gene encodes a
fucosyl transferase that adds a molecule of fucose with
α1–2 bond to the terminal galactose of the precursor oligosaccharide chain (therefore, fucose is the immunodominant sugar for the antigen H). The A allele encodes an
individuals agglutinated the red cells from both group A and group B
individuals. Serum of group AB individuals did not agglutinate group
A, B, and O RBC but red cells from group AB individuals were agglutinate from group A, B and O serum
N-acetyl-D- Galactosaminyltransferase, which adds, to
the terminal Galactose of substance H, a molecule of
N-acetyl-D- galactosamine with α1–3 bond (therefore,
the N-acetyl-D- galactosamine is the immunodominant
sugar for antigen A). The B allele encodes a galactosyltransferase, which adds a molecule of Galactose with a
α1–3 bond to the terminal Galactose of substance H
(therefore, Galactose is the immunodominant sugar for
antigen B). Individuals of group AB possess both a gene
A and a gene B and are therefore able to code for both
transferases and express both antigens (A and B). The
transformation of substance H into antigens A and B
reduces the serological reactivity of antigen H.The subjects of group O are double-dose carriers of a nonfunctional recessive gene, which is unable to code for any
glucosyltransferase. Substance H therefore remains
unchanged on the surface of the red cells. The very rare
subjects lacking the H gene are unable to modify the precursor oligopetide chain into H antigen. Consequently,
the transcripts of genes A and B, although present and
functioning, cannot carry out their action. These subjects
are defined as Bombay phenotypes. If, on the other hand,
the sese genotype is associated, these individuals do not
present substance A and B even in the secretions, while
substances A and B may be present in the secretions if a
Se gene is present.
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The polysaccharide chains that expose the immunodominant sugars H, A, or B can have a linear or multi-branched
form, the latter much more efcient in the exposure of the
antigen. At the molecular level, it is believed that the ancestral gene is gene A, from which gene B originated by mutation. The two genes differ for seven point mutations, four of
which result in an amino acid substitution (residues in positions 176, 235, 266, and 268). The O gene can result from
various mutations. Among these, the identication of a single nucleotide deletion that involves the insertion of a stop
codon, which then results in the translation of a truncated
protein devoid of transferase activity, is of particular
importance.
ABO blood group antigens are not fully developed at
birth, and their expression gradually increases to reach adult
life levels after 2–4years of life. It must also be remembered
that ABO antigens are ubiquitously expressed on the body’s
cells.
Numerous subgroups have been described within the
ABO system. The two main subgroups of A are called A1
and A2. On a molecular basis, they differ as the transferase
in A2 subjects is 22 amino acids longer than that of A1
subjects and less active than this in the elaboration of
highly repetitive and branched antigenic structures.
Subjects A1 and A2 (as well as subjects A1B and A2B) can
be differentiated by the use of lectins. They have been
described under weak groups of A (A3, Ax, Am, Ael).
Subgroups of B are much less frequent and of less practical importance.
Antibodies: Adult subjects normally have specic antibodies against antigen A or B, which is absent on its own
erythrocytes. These are IgM class immunoglobulins with a
wide thermal optimum (from 4 to 37 °C), able to x the
complement (therefore able to give hemolytic reaction after
transfusion), reactive in saline solution. It is believed that
the production of these antibodies (which are dened as
“natural,” since exposure to nonself-erythrocytes is not
demonstrable) derives from the fact that the congurations
that confer the antigenic specicities of determinants A and
B also exist on the bacterial walls of the germs constituting
the intestinal microbiota. At birth these antibodies are normally absent, while antibody production increases between
the fth and tenth year of life until it reaches adult levels and
decreases in the latter part of life. IgM represents the predominant immunoglobulin class of anti-A produced by
group B subjects and anti-B produced by group A subjects.
However, modest amounts of IgG class antibodies may still
be present in these subjects. IgG, on the other hand, is the
dominant antibody class in subjects of group O.Since IgG
is able to cross the placenta, infants of group A or B born to
mothers of group O may develop neonatal hemolytic disease, usually of modest entity. The serum of the subjects of
group O contains an antibody that is dened anti-A,B
because it reacts with both the A and B red blood cells. The
anti-A and anti-B reactivity of this antibody cannot be separated with selective absorption. It is not the sum of two antibodies (one anti-A specicity and one anti-B specicity),
but an anti-A,B cross-reactive antibody. For example, an
eluate prepared from group A red cells that have reacted
with anti-A, B will be reactive with both A and B cells alone.
Anti-A1 is detectable, as alloantibody, in a reduced percentage (<2%) in the serum of A2 subjects while it is more frequent (20–25%) in A2B subjects. This is usually a cold
antibody and is considered insignicant. Dolichos biorus
lectin preparations are available, which react with A1 but
not with A2 red cells.
H/h System
Group O red cells lack A and B antigens, and the membrane
expresses unchanged H antigen. In fact, the red blood cells of
group O subjects show a strong agglutination when tested
with the anti-H lectin obtained from Ulex europaeus
(Fig.16.3). The reaction observed is in descending order: O>
A2> B> A2B> A1> A1B.The rare subjects with the hh genotype whose red blood cells lack the H antigen (they do not
react with the anti-H lectin) at ABO typing are classied as
type O subjects but have, in addition to the anti-A and anti-B,
an alloantibody anti-H (therefore able to agglutinate normal
group O red blood cells). The term Oh Bombay is used to
designate subjects with this phenotype. At the genotypic
level, the Oh Bombay phenotype arises from the transmission of the hh alleles at the H locus and of sese at the Se
locus. Since the If allele is necessary for the formation of the
Leb antigen, the Oh red cells will, therefore, be Le (a + b–)
or Le (a– b–).
I/i System
Antigens: Antigens I and i are expressed on the erythrocyte
membrane on the same glycoproteins and glycosphingolipids that carry H, A, and B antigens and, in the secretions, on
the same glycoproteins that carry H, A, B, Lea, and Leb.
Nonetheless, antigens I and i are not antithetical, but are
expressed in temporal succession. At birth, neonatal red
blood cells are rich in i antigens, so for practical purposes,
cord blood cells are considered I−i +. During the rst 2 years
of life, the expression of antigen I gradually increases, while
that of antigen i decays. In adults, red blood cells are usually
highly reactive with anti-I and are considered I + i−. There is
a rare I−i + phenotype in adults. From a structural point of
view, antigen i appears to consist of a linear chain with at
least two repeating units Galactose-N-acetyl galactosamine,
joined by a β1–4 glycosidic bond. To the surface of the red
blood cells of adults, to give specicity I, these linear chains
are modied by the addition of branched structures consisting of N acetyl galactosamine that join the linear chains by
means of a β1–6 glycosidic bond.
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Galactoseminyltransferase
Fig. 16.3 ABO molecules. Alleles A, B, H encode for specic
Glycosyltransferases that add a specic sugar to a preexisting oligosaccharide chain. This additional carbohydrate determines the antigenic
specicity. These reactions occur sequentially: Therefore, the H gene
encodes a fucosyl transferase that adds a molecule of fucose with α1–2
bond to the terminal galactose of the precursor oligosaccharide chain
(therefore, fucose is the immunodominant sugar for the antigen H and
for group O individuals). The A allele encodes an N-acetyl-DGalactosaminyltransferase, which adds, to the terminal Galactose of
Antibodies: Anti-I and anti-i antibodies are often autoantibodies, are usually active in saline with thermal optimum
at 4°C, and are commonly identied with cold agglutinins,
which can take on clinical signicance if present at a titer
greater than 1/64 and, with a wide thermal range, are able to
x the complement. Anti-I auto antibodies are often produced by patients with Mycoplasma pneumoniae pneumonia. These patients may have transient hemolytic episodes
determined by the antibody. Patients with infectious mononucleosis often have anti-i antibodies.
Lewis System
Antigens: Antigens of the Lewis erythrocyte blood group
system are called Lea and Leb originate from the activity of
substance H, a molecule of N-acetyl-D-galactosamine with α1–3 bond
(therefore, the N-acetyl-D-galactosamine is the immunodominant sugar
for antigen A). The B allele encodes a galactosyltransferase which adds
a molecule of Galactose with a α1–3 bond to the terminal Galactose of
substance H (therefore, Galactose is the immunodominant sugar for
antigen B). Individuals of group AB possess both a gene A and a gene
B and are therefore able to code for both transferases and express both
antigens (A and B)
a glycosyltransferase encoded by the allele Le (or FUT3)
located on chromosome 19. This glycosyltransferase adds a
glucose residue to a precursor chain. The Lewis system
appears associated with the Sese system. In fact, the Lea
antigen is produced when the Le gene is inherited
together sese, and Leb is produced when it is inherited
together with Sese or SeSe. Therefore, Lea and Leb are not
antithetical antigens produced by alleles but depend on the
interaction of alleles inherited independently (Le, Se, se).
The antigens of the Lewis system are also not intrinsic to the
erythrocyte membrane, but are expressed on the Type 1 glycosphingolipid chains, which are adsorbed by the plasma on
the red cell membrane. Also in this case, four different phenotypes are possible, which present very different frequen-
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cies in the various ethnic groups: Le (a + b−) is present in
20% of the Caucasian and African population, Le (a−b +) is
present in 75% of Caucasians and 55% of black subjects, Le
(a−b−) is present in 5% of Caucasians and 25% of black
subjects, Le (a + b +) is rare in European populations and of
African ancestry, while it is common in Asian populations.
From a biochemical point of view, the synthesis of Lewis
antigens derives from the interaction of two different fucosyltransferases. One of them is produced by the Se locus and
one by the Le locus. Both enzymes act on the same substrate.
The fucosyltransferase encoded by the Le allele attaches a
molecule of fucose with α1–4 bond to the oligosaccharide
chain of the precursor. In the absence of the transferase
encoded by the Se allele, this conguration gives rise to the
Lea antigen. On the other hand, the Leb antigen is formed
when the fucosyltransferase encoded by the Se gene and
then, in sequence, the fucosyltransfease encoded by the Le
gene acts on the oligosaccharide chain of the precursor. In
this conguration, two fucose residues are added to the original oligosaccharide chain. The Leb antigen therefore reects
the presence of both the Le and Se alleles; while Lea antigen
reect the presence of Le without Se alleles. Lewis antigens
are rapidly adsorbed and eluted by the erythrocyte membrane. The transfused red blood cells lose their Lewis antigens and assume the recipient’s Lewis phenotype within a
few days of being released into the circulation.
Antibodies: Antibodies to Lea (more frequently) or Leb
(more rarely) are detectable, almost exclusively, in the sera
of Le (a−b−) subjects, usually in the absence of antigenic
stimulation evident from previous exposure to nonself red
blood cells. self. These are generally IgM antibodies.
Therefore, considering that Lewis antigens are also underdeveloped at birth, these antibodies are not associated with
MEN.Lewis antibodies can bind complement and fresh sera
containing anti-Lea can hemolyze incompatible red blood
cells invitro. Being IgM i, these antibodies react in saline
solution, forming rather fragile agglutinates that can be easily dispersed during too vigorous manipulation.
The genes that code for the antigens of the MNS system
are located on chromosome 4. The gene that codes for glycophorin A is called GYPA, the gene that codes for gly-
cophorin B is called GYPB. The two genes, which probably
derive from a single ancestral progenitor, have a homology
greater than 95%. Glycophorin A is a transmembrane glycoprotein present in approximately 1,000,000 copies for
each erythrocyte. The M and N antigens are located in an
extracellular portion consisting of 72 amino acid residues
linked to side glucose chains. M specicity is characterized by a serine in position 1 and a glycine in position 5;
the specicity N is characterized by a leucine in position 1
and a glutamate in position 5. Glycophorin B is smaller
and is present in about 200,000 copies for each erythrocyte. The specicity S is characterized by a methionine in
position 29, this position is occupied by a threonine to give
the specicity s. Usually the M and N antigens are
destroyed by treatment with proteolytic enzymes such as
cin or papain.
Antibodies: Anti-M is frequently detected as a cold reactive agglutinin with red cells suspended in physiological
solution. These antibodies are usually of the IgM class
(although the nding of IgG is not uncommon) and are frequently observed in the serum of subjects who have never
been exposed to nonself human erythrocytes. These are antibodies that only rarely take on clinical signicance, although
they have been implicated in rare cases of PTH and
HDFN.These were mostly IgG class antibodies able to react
at 37°C or with the antiglobulin test.
Anti-N is a relatively rare antibody, it is usually of the
IgM class, and behaves like a cold agglutinin. They are not
considered clinically signicant antibodies. The “anti-S” and
“anti-s” antibodies are usually immune antibodies of the IgG
class, identiable by the antiglobulin test. They are considered clinically signicant, being implicated in PTH and
HDFN.Anti-U is rare but should be considered if a reactive
antibody against a high frequency antigen is identied in a
subject of African origin previously exposed to nonself red
blood cells.
MNSs System
Antigens: It is a complex system, consisting of over 40 glycoproteic antigens. The M, N, S, s antigens together with U
(a high-frequency antigen) are the most relevant in
Transfusion Medicine. The M and N antigens are localized
on glycophorin A; S, s, and U are located on glycophorin
B.The genes encoding these antigens exhibit marked linkage disequilibrium. As an example, the Ns gene complex is
more frequent than NS. The MNS system also includes
numerous low-frequency antigens, resulting from mutations
involving amino acid substitutions and from modications in
glycation.
P System
Antigens: The P group system historically included the antigens P, P1, Pk and Luke. However, further studies have claried how two biosynthetic pathways and genes located in
different loci are involved in the development and expression
of these antigens. At present, according to the ISBT nomenclature, the P antigen is part of the GLOB system; P1 remains
assigned to the P system, while Pk and Luke are included in
the GLOB29 antigenic collection. The antigens of the P system, similarly to those of the ABO system, are synthesized
sequentially by adding single glucose molecules to “precur-
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sor” oligosaccharide chains. These antigens are expressed on
glycolipid type molecules. In the case of the P blood group
system, the common precursor is lactosylceramide. This
molecule, by means of two metabolic pathways, is converted
to P1 antigens or to antigens of the globoside series (Pk, P or
LKE). There are two common phenotypes associated with
the P system (P1 and P2) and three rare phenotypes (p, P1k
and P2k). The P1 gene is located on chromosome 22, while
the P gene is located on chromosome 3. The P antigen has
been shown to be a receptor for erythrovirus (parvovirus)
B19. Therefore, subjects with the p phenotype (lacking the
globoside antigen) appear naturally resistant to infection.
Antibodies: The serum of P1 negative subjects often contains an anti-P1. It is, therefore, as a rule, a natural antibody,
of the IgM class, which reacts optimally at +22°C.It is not
thus able to determine MEN and, being only rarely implicated in PTH, it is considered to be of little signicance from
a clinical point of view. The expression of the P1 antigen on
the surface of the red cells can be very variable and tends to
decrease with their conservation. This can lead to difculties
in identifying the antibody specicity. P1k and P2 subjects
may develop an anti-P antibody. It is a natural antibody, consisting of a mixture of IgM and IgG, characterized by a wide
thermal range (reacts from 4 to 37°C), considered clinically
signicant in how much can be associated with PTH and,
although more rarely, with HDFN.The anti-P autoantibody
has also been associated with paroxysmal cold hemoglobinuria; it is a cold IgG autoantibody which is also described as
biphasic hemolysis.
RhD RhCE System
As part of the numerous nomenclature and classication proposals of the Rh system antigens, in this paragraph the Fisher
and Race nomenclature will be used, updated according to
current genetic and biochemical knowledge. Historically, the
Rh antigen (later identied with the D antigen) was described
in 1939 by Levine in a woman whose infant was found to
have HDFN, and she herself developed a hemolytic posttransfusion reaction after transfusion of red blood cells
donated by her husband. The name Rh derives from the fact
that in 1940, Landsteiner and Wiener described an antiserum
obtained by immunizing guinea pigs with Macaques rhesus
monkey red cells, capable of reacting with 85% of human
red blood cell samples, and which behaved like the antisera
obtained by women whose foetuses had suffered from
MEN.Later, in the second half of the 1940s, four additional
antigens named C, E, c and e were included in the Rh system,
which currently consists of more than 50 antigens, thus
becoming the much larger erythrocyte blood group system.
Antigens of the Rh system begin to be represented on the
surface of the red cells as early as the eighth gestational
week, and are fully expressed in the term new-born. They are
characteristic exclusively of red blood cells, not being
expressed in other cells or tissues.
Genes: At present it is believed that the antigens of the
Rh system (D, C/c, E/e) are encoded by two alleles called
RHD and RHCE, located on chromosome 1in two contiguous and closely linked loci. These alleles encode nonglycosylated polypeptides expressing Rh antigens. The RHD
gene encodes a transmembrane protein expressing antigen
specicity D, while a d antigen has never been described. In
D-negative subjects of Caucasian origin the RHD gene is
deleted, while in subjects of African or Asian origin the
D-negative phenotype is frequently associated with an inactive or mutated RHD gene. The RHCE gene encodes a single
nonglycosylated polypeptide, which expresses the certain
antigenic C, c, E, and e (its alleles are RHCe, RHCE, RHcE,
and RHce). From a biochemical point of view, both the RHD
gene and the RHCE gene encode a transmembrane protein
of 417 amino acid residues. Unlike what has been observed
for other antigenic determinants of erythrocyte blood group
systems, this polypeptide chain does not present glycosylation but bonds fatty acids. There is a high homology
between the products of the different alleles of the RHCE
gene. For example, the C and c antigens differ from each
other only by four amino acids in positions 16, 60, 68 and
103. The presence of a proline or an alanine in position 226,
on the other hand, differentiates the E allele from that e. Rh
proteins (D and CE) are complexed with Rh-associated lipoprotein (RhAG), which has 37% homology with Rh polypeptides, and is encoded by the RHAG gene located on
chromosome 6.
Rh phenotype and genotype: commercial anti-D, anti-C,
anti-E, anti-c and anti-e antisera are used for the determination of the Rh phenotype. The set of antigens detected on a
subject’s red blood cells therefore constitutes its Rh phenotype. D negative subjects lack the RHD gene that encodes the
D antigen or have a nonfunctional RHD gene. Most of the
negative D subjects are homozygous for the RHce allele,
which represents the gene encoding the antigens c and e.
Less frequently they may have RHCe or RHcE alleles, which
code for C and e or for c and E, respectively. The RHCE
gene, which produces the C and E antigens, is very rare in D
negative individuals. The D genotype of D positive persons
cannot be determined with serological tests, as molecular
biology techniques must be used.
In Rh system, the interaction between genes (position
effect) is well described. If the interaction occurs between
genes located on the same chromosome, it is referred to as
the cis effect; if it occurs between genes placed on homologous chromosomes, it is indicated as a trans effect. In example (cis effect), the E antigen produced by DcE appears
quantitatively less expressed than the E antigen produced by
cE.It was also observed that both C and E were less expressed
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when they were encoded by the DCe/DcE genotype than the
antigens encoded by DCe/ce or DcE/ce (trans effect).
Usually positive D red cells show, when tested with anti D antiserum, strong agglutination, appreciable macroscopically, which allows rapid and safe classication. The
classication of red blood cells that do not agglutinate after
being tested with an anti-D is not so simple. Reactivity can in
fact be reactive-dependent or can only be revealed by the
antiglobulin test. These “weak” Ds (formerly called Du) are
determined in most cases by an RHD gene that encodes a
RhD protein with reduced quantitative expression of the D
antigen on the erythrocyte membrane. This phenotype is
quite common in populations of African origin. In example,
erythrocytes of some subjects with the Dce/Ce genotype
show a weak D antigenic expression due to a suppressor
effect exerted by RHC in the trans position with respect to
RHD. Moreover, it is wellknown that the D antigen consists
of numerous distinct structural components; RBCs that lack
some components of the D antigen are referred to as “partial
D” and, especially those with the DVI phenotype, can react
only with some anti-D monoclonal antisera and can produce
anti-D antibodies. There are many other antigens belonging
to the Rh erythrocyte blood group system. Only some of
them, such as Cw (antithetic to C), the G antigen (characterized by a serine in position 103 of the Rh polypeptide), the f
antigen (a ce fusion antigen), have practical importance.
Very rare family groups are also described, whose red blood
cells appear to be devoid of all Rh (Rh null) antigens. The Rh
null phenotype can be of the “regulatory” type and therefore
derive from a mutation of the RHAG gene, or of the “amor-
phous” type because of mutations of RHCE genes associated
with deletion of the RHD gene.
Antibodies: Usually the antibodies directed towards antigens of the Rh system are of the immune type and derive from
exposure to nonself red blood cells. It is therefore IgG with
thermal optimum at 37°C, which react with enhancing media
or with the antiglobulin test. In some cases, the anti- Rh antibodies may be natural (for example, anti-E or anti- Cw). As
regards the ability to induce the production of antibodies after
stimulation, antigen D is undoubtedly the most powerful
immunogen, followed in descending order by antigens c, E, C,
and e. It must be remembered that individuals with partial D
may develop an anti-D alloantibody. Rh antibodies do not generally x complement. They therefore result in extra-vascular
hemolysis invivo. They are considered clinically signicant,
as they are potentially associated with even severe HDFN and
posttransfusion hemolysis (mainly extra-vascular).
Landsteiner–Wiener (LW) System
The Antigens: The Landsteiner–Wiener (LW) blood group
system, although constituting an independent system,
appears to be linked to the Rh system. In fact, it is the antigen
identied by the original anti-Macaques rhesus serum used
to dene positive D subjects. Four antigens are known; to the
original LW1 (present in the D + subjects) and LW2 (present
in the D− subjects), further antigens named LW3 and LW4
were associated. The genes encoding the antigens of the LW
system are located on chromosome 19.
Antibodies: The antibodies of the LW system are not considered clinically signicant, as they are unable to generate
MEN and PTH.
Lutheran System
Antigens: Lutheran erythrocyte blood group system antigens
are named Lua and Lub. They are encoded by two codominant alleles in the LU gene, located on chromosome 19. Four
phenotypes are therefore possible: Lu (a+b−) present in less
than 1% of the population, Lu (a+ b+) present in 7.5%, Lu
(a−b +) present in 92%, and Lu (a−b−) very rare. A series of
high-frequency antigens (Lu4, Lu5, Lu6, Lu7, Lu8, Lu11,
Lu12, Lu13, Lu16, Lu17, and Lu20) and two low frequency
antigens (Lu9 and Lu14) were included in the Lutheran system. Antigens of the Lutheran system are carried by a glycoprotein that appears to play a role in cell adhesion. The
antigens of the Lutheran system are poorly expressed on the
surface of foetal red blood cells, and the expressiveness
increases during life, while remaining all in all modest (from
500 to 4000 molecules per erythrocyte, depending on the
phenotype).
Antibodies: These are antibodies that are rarely found.
They are largely produced in response to pregnancy or
transfusions but have also been detected in the absence of
obvious exposure to nonself red blood cells. The anti-Lua
antibody is not considered clinically signicant, as it is not
associated with PTH or HDFN.The anti-Lub antibody was
associated with moderate HDFN and reduced survival of
the transfused red cells, but not with PTH.Most anti-Lua
and some anti- Lub antibodies directly agglutinate red blood
cells in saline, producing a typical mixed-eld reactivity
pattern, with small agglutinates scattered among most free
red blood cells.
Kell/Cellano System
Antigens: The major antigens of the system are the Kell
(K) antigen present in 9% of Caucasians and 2% of blacks,
and the cell (k) antigen present in over 99% of individuals.
Other antigens of the Kell system, antithetical to each
other, are Kpa/Kpb, and Kpc; Jsa/Jsb; K11/K17; K14/K24.
An ethnic distribution is also observed for the Kpa and Jsa
antigens, as Kpa is mainly observed in white subjects
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while Jsa is predominantly detected in subjects of African
origin. Also described are subjects totally devoid of antigens referable to the Kell blood group system, dened as
Ko or Knull. The antigens of the Kell system are located
on a 93-kD transmembrane protein rich in cysteine and
capable of forming sulfhydryl bonds. They are, therefore,
conformational antigens, sensitive to the treatment of red
blood cells with agents capable of altering the secondary
structure of the molecule (mercaptoethanol, for example).
It appears that this protein belongs to the family of neutral
endopeptidases, which act as a bond for Zn; it also has a
high similarity with the common antigen of lymphoblastic
leukaemia (CALLA or CD10). Although the Kell system
locus is located on chromosome 7 and the Kx (XK) locus
is located on the X chromosome, it appears that the K and
Kx proteins form a covalent complex on the surface of the
red cells. The lack of Kx antigen is accompanied not only
by a weak expression of the Kell antigens, but also by
acanthocytosis and reduced erythrocyte survival. These
erythrocyte abnormalities can be accompanied by an
increase in the creatine kinase (CK) enzyme and neuromuscular abnormalities, constituting the so-called McLeod
phenotype, probably linked to abnormalities of the XK
locus.
Antibodies: The K antigen is highly immunogenic. Anti-K
antibodies are, therefore, frequently detected in the serum of
transfused kk patients, although anti-K have rarely been
observed even in subjects never exposed to nonself erythrocytes. These are IgG class immunoglobulins, with an optimum temperature at 37 °C, reactive with the antiglobulin
test. Being able to generate PTH and HDFN, they are considered clinically signicant antibodies. The k antigen is also
highly immunogenic, as it can generate the appearance of
IgG-class allo-antibodies, with an optimum temperature of
37°C, reactive with the antiglobulin test. Being able to generate PTH and HDFN, they are also considered clinically
signicant. Anti-Kpa, anti-Kpb, Anti-Jsa, and anti-Jsb antibodies are all much less frequent than anti-K, but have similar serological characteristics, and are therefore to be
considered clinically signicant.
Duy System
Antigens: The antigens of the Duffy erythrocyte blood
group system, called Fya and Fyb, are encoded by a pair
of co- dominant alleles in the FY locus, located on chromosome 1. As these are co-dominant alleles, four phenotypes are possible: Fy (a+b−), Fy (a+b+), Fy (a–b+) and
Fy (a–b–). In Caucasians, individuals with the Fy (a−b−)
phenotype are extremely rare, while the frequency can be
as high as 68% among individuals of African descent.
From the biochemical point of view, the Fya and Fyb
antigens are localized in the N-terminal fraction of transmembrane glycoproteins and are, therefore, eliminated
by treatment with proteolytic enzymes. The above antigens have ubiquitous diffusion. In addition to the surface
of the red cells, they are present in other tissues such as
central nervous system, spleen, kidney, lung. Glycoprotein
constitutes the receptor for the malarial parasite
Plasmodium vivax; people with red blood cells lacking
the Fya and Fyb antigens are therefore resistant to this
infection.
Antibodies: Both anti-Fya and anti-Fyb antibodies are
usually IgG class immunoglobulins, with thermal optimum
at 37°C reactive with the antiglobulin test. The anti-Fya antibody is quite common and is considered clinically signicant as it is capable of generating PTH and HDFN.Anti-Fyb
is a rather infrequent antibody, and only rarely implicated in
PTH and HDFN.Weak anti-Fya and anti-Fyb often express
a dose-dependent effect.
Kidd System
Antigens: The antigens of the Kidd erythrocyte blood group
system, named Jka and Jkb, are encoded by two codominant
alleles in the HUT11 gene located on chromosome 18. Four
phenotypes are therefore possible: Jk (a+b−), Jk (a+ b+), Jk
(a−b+), and Jk (a−b−), the latter of which is extremely rare.
From a biochemical point of view, the antigens of the Kidd
system are expressed by an erythrocyte membrane protein
involved in urea transport.
Antibodies: Both anti-Jka and anti-Jkb antibodies are
usually IgG class immunoglobulins, with thermal optimum at 37°C, reactive at the antiglobulin test. Both are
considered clinically signicant, being able to generate
PTH and HDFN. Characteristic of anti-Jka and anti-Jkb
antibodies is their ability to produce severe delayed posttransfusion hemolytic reactions. In these subjects, it is
possible to highlight a previous exposure to the antigen,
which resulted in immunization with a modest antibody
titer and not detectable with routine use tests. Nonetheless,
in response to a new exposure to the antigen, an “anamnestic” antibody response rapidly develops against antigens of
transfused red blood cells, which are destroyed when they
are still present in the circulation. Both anti-Jka and antiJkb are often weakly reactive, with a score that tends to
decrease depending on the conservation of the sample.
They are more easily detectable in the presence of complement if a polyspecic Coombs serum with anti-complementary activity is used. Alternatively, the identication of
reactivity can be improved by using a two- stage antiglobulin test or enzyme treatment. Both antibodies are more easily detectable using homozygous red blood cells
(dose-dependent effect).
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