Blood Grouping Systems and Typing Techniques
A blood group system
consists of antigens produced, either directly or indirectly, by alleles
located at a single genetic focus or at loci so closely linked that
crossing-over between the loci rarely occurs.(1) Inheritance of an
allele usually, but not always, leads to the appearance its corresponding
antigen on RBC membranes or in body secretions. Variant forms of a given
allele can be inherited that produce increased or depressed amounts of the
corresponding antigen. Antigen expression can also be influenced by genes
inherited independently other blood group loci. For example, the
expression of A or B antigens is affected by the action of the I gene or
by alleles at the H blood group locus.
ABO, H, P, I or Lewis
blood group antigens are constructed on structurally related carbohydrate
molecules through the activity of gene-encoded glycosyltransferase
enzymes. Since it is possible for the carbohydrate molecules to carry the
determinants of more than one of these blood groups, the activities of the
genes of one of these blood groups may affect the expression of the
antigens of another. The antigens appear when specific sugars are added by
the transferases to the ends of carbohydrate chains called
oligosaccharides, The sugars added to the precursor chains by the gene
transferases are referred to as immunodominant sugars since the sugars
confer specific antigenic activity to the terminal portions of the
converted oligosaccharide chains .(2)
consist of chains of sugars that can be attached to either glycoprotein or
glycosphingolipid carrier molecules. In glycoproteins, oligosaccharides
are linked via galactosamine (GalNAc) to polypeptide backbones.
Structurally similar oligosaccharides are attached via glucose (Glc) to
ceramide residues in glycosphingolipids. Glycosphingolipids form part of
RBC membranes and also the membranes of most endothelial cells. Soluble
forms are present in plasma, but are not secreted in body fluids. Soluble
glycoproteins with blood group antigen activity exist in the body's serous
and mucous secretions. Membrane-associated glycoproteins are present
on RBCs and other body cells. (3)
The ABO System
A series of tests
performed by Karl Landsteiner in 1900 led to the discovery of the ABO
blood groups and to the development of routine blood grouping procedures.
Landsteiner tested blood samples from his colleagues by mixing each
person's serum with suspensions of RBCs from the others. Noting
agglutination in some mixtures, but not in others, he was able to classify
the blood samples into one of three groups, now named A, B and 0. (The
fourth group, AB, was discovered in 1902 by Landsteiner's pupils von
Decastello and Sturh.) Landsteiner recognized that the presence or absence
of only two antigens, A and B, was sufficient to explain the three blood
groups he saw. He also showed the serum of each person contained an
antibody directed against the antigen absent from that person's RBCs. The
first blood group system to be discovered, the ABO blood groups remain the
most significant for transfusion practice. It is the only system in which
the reciprocal antibodies are consistently and predictably present in the
sera of normal people whose RBCs lack the corresponding
ABO incompatibility between a recipient and the donor is the
foundation on which all other pretransfusion testing rests.(5.6)
Biochemical and Genetic Considerations
carrying A or B oligosaccharides are integral parts of the membranes of
RBCs, epithelial and endothelial cells; they are also
present in soluble form in plasma. Glycoproteins that carry identical
oligosaccharides are responsible for the A and B activity of secreted body
fluids such as saliva. A and B oligosaccharides that lack carrier protein
or lipid molecules are found in milk and urine.
Genes at three separate
loci (ABO, Hh, and Sese) control the occurrence and the location of the A
and B antigens. Three common alleles --A, B and O-are
located at the ABO locus on chromosome 9. The A and B genes encode
glycosyltransferases that produce the A and B antigens, respectively.(7)
The O gene is considered to be amorphic since no detectable blood group
antigen results from its action. The RBCs of group O persons lack A and B,
but carry an abundant amount of H antigen because this antigen is the
precursor material on which A and B antigens are built.
Family studies have
shown that the genes at the remaining two loci, Hh and Sese (secretor),
are closely linked. The chromosome on which they are located has not yet
been identified. It is suggested that one of these loci may have arisen
through gene duplication of the other. (8) Two recognized alleles reside
at each locus. Of the two alleles at the H locus, one of these, H,
produces an enzyme that acts at the cellular level to construct the
antigen on which A or B are built. The other allele at this locus, h, is
very rare. No antigenic product has been linked to h, so this gene is also
considered an amorph. The possibility exists that other alleles occur at
the Hh locus that differ from H in that they cause the production of only
very small amounts of H antigen. (9)
The Se gene is directly
responsible for the expression of H (and indirectly responsible for the
expression of A and B) on the glycoproteins in epithelial secretions such
as saliva. Eighty percent of the population are secretors because they
have inherited the Se gene and produce H in their secretions that can be
converted to A and/or B (depending on the genetic background of the
secretor). The se gene, having no demonstrable product, is an amorph.
on which the A and B antigens are built can exist as simple structures of
a few sugar molecules linked together in linear fashion. They can also
exist as more complex structures that are composed of many sugar residues
connected together in branching chains. It has been proposed that the
differences in cellular A, B and H activity seen between specimens from
infants and adults may be related to the number of branched structures
carried on the cellular membranes of each group.(10) The RBCs of infants
are thought to carry A, B and H antigens built predominantly on linear
oligosaccharides. Linear oligosaccharides have only one terminus to which
the H, then A and B, sugars can be added. In contrast, the RBCs of adults
appear to carry a high proportion of branched oligosaccharides. Branching
creates additional portions on the oligosaccharide that can be converted
to H and then to A and B antigens.
A and B genes do
not produce antigens directly but instead produce enzymes called
glycosyltransferases that add specific sugars to oligosaccharide chains
that have been converted to H by the action of the H gene. H antigens are
constructed on precursor oligosaccharide chain endings called Type 1 and
Type 2. (2.7) The number 1
carbon of the terminal 6-carbon sugar b-D-galactose
(Gal) is linked to the number 3 carbon of subterminal
N-acetyl-glucosamine (GluNAc) in Type 1 chains and to the
number 4 carbon of GluNAc in Type 2 chains. Blood group-active
glycoproteins present on cell surfaces or in body fluids carry either Type
1 or Type 2 chains. Glycosphingolipids present in the plasma and those on
the membranes of most glandular and parenchymal cells also have either
Type 1 or Type 2 chain endings. In contrast, the glycolipid antigens
produced by the RBCs; appear to be formed exclusively of Type 2 chains.
These chains are carried on a class of glycosphingolipids called
At the cellular level,
the H gene transferase produces a fucosyltransferase that adds fucose (Fuc)
in alpha (1-2) linkage to the terminal Gal of Type 2 chains. The A
and B gene transferases can only attach their immunodominant sugars when
the Type 2 (or Type 1) chains have been substituted with Fuc (ie, changed
to H) thus, the A and B antigens are constructed at the expense of H. The
A gene-specified N-acetyl-galactosaminyl-transferase
and the B gene-specified galactosaminyl-transferase add GalNAc
and Gal, respectively in alpha (1-3) linkages to the same Gal acted
on by the H gene transferase.
alleles at the ABO locus that result in subgroups (phenotypes of A and B
that differ from each other with respect to the amount of A or B carried
on the RBCs) produce transferases that differ from one another in their
ability to convert H antigen .(7,11)
The O gene is thought to produce a protein that can be detected
immunologically but has no detectable transferase activity. As a
consequence, the RBCs of group O persons carry readily detectable,
unconverted H antigen. The secretion of Sese persons contain Type I and
Type 2 chains with no H, A or B activity. It has been suggested that the H
and Se genes each encode a different
fucosyltransferase. (8,11) The enzyme produced by H acts primarily
on Type 2 chains and in RBC membranes. That produced by Se prefers (but
does not limit its action to) Type 1 chains and acts primarily in the
secretory. Studies performed on the secretions of persons with the rare Oh
phenotype support the concept that two types of H antigen exist.(9)
Persons of this phenotype,
who are genetically Hh and Sese, have no H and therefore, no A or B
antigens on their RBCs or in their secretions. However, H, A and B
antigens are found in the secretions of genetically hh persons, who,
through family studies, appear to possess at least one Se gene.
and B antigens are detected in direct agglutination tests with
anti-A and anti-B reagents. ABO reagents frequently produce weaker
reactions with the RBCs of newborns than with RBCs from adults. Weaker
reactions are encountered because A and B antigens are not fully developed
at birth, even though they can be detected on the RBCs of embryos
5-6 weeks old. (5-13) By
the time a person is 2-4 years old, RBC A and/or B antigen
expression is fully developed. Antigenic expression remains fairly
constant throughout life, although decreases have been seen in old age.
Subgroups of A
of A are phenotypes that differ from others of the same ABO group with
respect to the amount of A antigen carried on RBCs, and, in
secretors, present in the saliva.
The two principal subgroups of A are A1, and A2. RBCs of both react
strongly with anti A reagents in direct agglutination tests. The
serologic distinction between A1, and A2 is based on results obtained in
tests with reagent anti-A1, prepared from group B human serum or the
lectin of Dolichos biflorus seeds. Under prescribed testing conditions,
anti-A1, reagents agglutinate A1, but not A2 RBCs. The RBCs of
approximately 80% of group A or group AB persons are agglutinated by
anti-A1, and, thus are classified as A1, or A1B. The remaining 20%
whose RBCs are agglutinated by anti-A, but not by anti-A1,
are called A2, or A2B. (16)
Anti-A1 occurs in
the serum of 1% to 8% of A2 persons and 22% to 35% of A2B persons.(13)
Anti-A1 can cause discrepancies in ABO testing and
incompatibilities in crossmatches with A1, or A1B RBCs. It is considered
to be clinically insignificant unless it reacts at 37 C. It is not
necessary to test group A RBCs with anti A1, to confirm their subgroup
status except when working with samples from people whose sera contain
Subgroups weaker than
A2 occur infrequently and, in general, are characterized by decreasing
numbers of A antigen sites on the red cells and a reciprocal increase in H
antigen activity. The genes responsible constitute less than 1% of the
total pool of A genes. Classification of weak subgroups is generally based
Degree of RBC agglutination by anti-A and anti-A1.
Degree of RBC agglutination by anti A, B.
Degree of H reactivity of the
Presence or absence of anti-A1 in the serum.
Presence of A and H substances in the saliva of secretors.
RBCs of the Ax Ael,
Aint or A3 subgroups are seen only infrequently in transfusion practice.
Ax and Ael RBCs are readily recognized as subgroups of A by the
discrepancies they produce between RBC and serum grouping tests. Ax RBCs
are, in general, agglutinated by human anti-A,B but not by human
anti-A. However, Ax RBCs react with some murine monoclonal
anti-A reagents.(15) Ael RBCs fail to react with anti-A or
anti-A,B of any origin. Adsorption and elution studies are necessary
to show that these RBCs carry the A antigen. RBCs of the Aint phenotype
can be identified only if tests with anti-A1 are performed. Aint
RBCs react more weakly than A1 RBCs with anti-A1, yet more strongly
with anti-H than do A2 RBCs. A3 RBCs produce a characteristic
mixed-field pattern of small agglutinates among many free RBCs in
tests with anti-A and anti-A,B. Weak subgroups of A such as
Ax, Ael, and Aint, cannot be identified on the basis of blood grouping
tests alone. Saliva studies and adsorption/elution studies must be
Subgroups of B are even
less common than subgroups of A.
Antibodies to A and
possess antibodies directed toward the A or B antigen absent from their
own RBCs. This predictable complimentary
relationship permits serum grouping in addition to RBC ABO grouping tests
. The immunoreactive configurations that confer A
and B specificities to molecules of the RBC membrane also exist in other
biologic entities, notably bacterial cell walls. Bacteria are widespread
in the environment and it appears that their presence in intestinal flora,
dust, food and other widely distributed agents ensure a constant exposure
of all persons to A-like and B-like antigens. Immunocompetent
persons react to the environmental antigens by producing antibodies to
those that are absent in their own systems. Thus, anti-A occurs in
the sera of group O and group B persons and anti-B occurs in the
sera of group O and group A persons. Group AB people, having both
antigens, make neither antibody.
Time of Appearance
anti-B production generally begins after the first few months of
life. Occasionally infants can be found that are already producing these
antibodies at the time of birth. (5) Antibody production remains fairly
constant until late in adult life. In elderly people, anti-A and
anti-B levels may be lower than those seen in young adults. (1,16)
Since antibody production normally begins after birth, results that
are obtained with the sera of newborns or infants to about 4-6
months cannot be considered valid because the antibodies may have been
acquired through the placental transfer of maternal IgG anti-A and
Reactivity of Anti-A and
Anti-A produced by group B persons and anti-B produced by A
people are composed predominantly of IgM molecules.(5) Small quantifies of
IgG molecules are also present in the sera of these two groups. IgG is the
dominant form of anti-A and anti-B of group O serum. The IgG forms
readily cross the placenta and can cause ABO hemolytic disease of the
newborn (HDN). Because of the predominance of IgM antibodies in the sera
of group A or B persons, ABO hemolytic disease is rarely seen in ABO-incompatible
infants born of group A or B mothers. (5,13)
features of IgM and IgG anti-A and anti-B are given in Table
10-3. Both immunoglobulin types preferentially agglutinate red cells
at room temperature (20-25 C) or below. Both are efficient
activators of complement at 37 C. The complement-mediated lytic
capabilities of these antibodies are most apparent when an incubation
phase at 37 C is added to serum grouping tests. Occasionally, patients or
donors can be found whose sera cause the hemolysis of ABO-incompatible
red cells at temperatures below 37 C. Hemolysis by ABO antibodies in serum
grouping tests should be suspected when a pink to red discoloration
appears in the supernates or when the buttons of reagent ABO grouping RBCs
are reduced in size or are missing. Hemolysis must be interpreted as a
positive result. Hemolysis of RBCs will not occur if reagent RBCs are
suspended in solutions that contain EDTA or other anticoagulants that
prohibit complement activation.
Agglutinin development and cause
“It is difficult to
understand how agglutinins are produced in individuals who do not have the
respective antigenic substances in their red blood cells. However, small
amounts of group A and B antigens are believed to enter the body in the
food, in bacteria, or by other means, and these substances presumably
initiate the development of anti-A or anti-B agglutinins.”
-Guyton, Textbook of
Anti-A,B (Group O Serum)
Serum from group O
persons contains an antibody designated as anti-A,B. It reacts with
A and B RBCs and activities for both RBC groups cannot be separated by
differential adsorption. Eluates prepared from group A RBCs that have been
used to adsorb group O serum contain anti-A and an antibody that
reacts with both A and B RBCs. Similar findings are obtained when B RBCs
are used for adsorption. Saliva from A or B secretors inhibits the
activity of this antibody with either A or B RBCs.
The anti-A of
group B serum appears, from simple studies, to contain separable
anti-A and anti-A1. In direct tests, group B serum
agglutinates A1 and A2 RBCs, yet following adsorption with A2 RBCs, group
B serum reacts only with A1 RBCs. If further tests are performed, the
differences between A antigen expression on A1 and A2 RBCs appears to be
quantitative rather than qualitative. Further adsorption of group B serum
with A2 RBCs will remove all serum activity for A1 RBCs. The apparent
anti-A1 made by adsorption of group B serum can be thought of as a
weakened form of anti-A. It reacts with A1 RBCs because they have
more A antigen than do A2 RBCs. The sera of persons of certain weak
subgroups of A may contain anti-A1 that is serologically similar to the
anti-A1 of group B adsorbed serum.
Adsorbed group B serum
can be used at the practical level to differentiate the two common A
subgroups. More frequently, however, anti-A, reagents are employed
that are manufactured from the lectin of Dolichos biflorus. The lectin
will react with A, and A2RBCs unless it has been diluted appropriately.
Reagent anti-A, lectins have been diluted by the manufacturer to
react with & but not A2, RBCs.
Routine Testing for ABO
RBC typing tests, using
anti-A and anti-B to determine the presence or absence of the
antigens, are often referred to as direct or forward grouping tests. Serum
grouping tests, using reagent A, and B RBCs to detect serum anti-A
and anti-B, are sometimes called reverse grouping tests. Routine
grouping of donors and patients must include both RBC and serum tests,
each serving as a check on the other. It is permissible to test RBCs only
when ABO grouping is performed to confirm the group of donor blood that
has already been labeled with a blood group designation or when testing is
performed on samples from infants less than 6 months of age.
Some ABO RBC grouping
reagents are prepared from pools of sera from persons who have been
stimulated with A or B blood group substances to produce antibodies of
high titer. Other ABO grouping reagents are manufactured from monoclonal
antibodies derived from cultured cell lines. Both types of reagents are
potent and agglutinate most antigen positive RBCs on direct contact
without centrifugation. Serum testing is most reliably performed by tube
or microplate methods. Anti-A and anti-B occurring in the sera
of patients and donors are frequently too weak to agglutinate RBCs without
centrifugation. Therefore, it is not recommended that serum grouping tests
be performed on slides.
On occasion, other
reagents are incorporated into ABO grouping procedures. These include
anti-AB (RBC grouping) and reagent A2 and O RBCs (serum grouping).
Anti-A,B reagents, such as anti-A and anti-A,B, are
derived either from human sera or monoclonal cell lines. Some workers
elect to use anti-A,B routinely in grouping tests to avoid
mistakenly classifying weakly reactive A or B RBCs as group O.
Unfortunately, there exists a misconception that anti-A,B is more
potent than either anti-A or anti-B and thus, will detect most
weak subgroups of A or B. With the exception of Ax, anti-A,B (particularly
that of human origin) does not agglutinate the RBCs of less common
subgroups that fail to react with anti-A or anti-B. (13)
Human-source anti-A,B does not react well with Ax RBCs in
immediate-spin tests. The reagent and RBCs must be incubated
together for 1060 minutes at room temperature for reactions to occur. If
the manufacturer's directions recommend using anti-A,B for the
detection of weak subgroups, it means that its reactivity against A. RBCs
has been demonstrated. AABB Standards
does not require the use of anti-A,B to detect weak A or B
subgroups since such bloods often distinguish themselves from O by failing
to produce the expected serum grouping results. Moreover, the adverse
consequences associated with the transfusion of weak subgroups of A and B
to group O recipients are minimal.
prepared reverse grouping reagents contain A2 and O RBCs in addition to A,
and B RBCs. The sole purpose of A2 RBCs in these reagents is to facilitate
the recognition of anti-A, in subgroups of A
Since the majority of A blood does not contain anti-A2, many
workers employ this reagent only when discrepancies between RBC and serum
tests are encountered. Group O RBCs of reverse grouping sets can be used
to identify those sera that contain cold-reactive agglutinins that
may interfere with serum grouping tests. Generally, such RBCs cannot be
used for the detection or identification of unexpected antibodies since
they have not been manufactured to meet the requirements of the FDA for
Manufacturers of ABO
reagents provide, with each reagent package, detailed instructions for the
use of the reagent. Instructions may vary from one manufacturer to another
in testing requirements. Therefore, it is important to follow the
directions supplied with the specific reagent in use.
discrepancies can be traced to problems arising in RBC grouping tests.
Samples obtained from patients who have received transfusions
recently or who have received a bone marrow transplant may produce
unexpected reactions if the samples contain a mixture of RBCs that differ
from each other in their ABO group (transfusion or transplantation
Blood samples from persons who have inherited variant A or B genes
may carry poorly expressed antigens. Weak antigens are also found on the
red cells of some people with diseases such as leukemia. (1,5) Samples
from these people may fail to produce the expected reactions in direct
agglutination tests with anti-A and anti-B.
Abnormalities of an inherited or acquired nature, leading to what
is referred to as polyagglutinable states, can result in RBCs with
modified membranes. The modified RBCs can be unexpectedly agglutinated by
reagent anti-A, anti-B or both.
Abnormal concentrations of serum proteins, the presence of
macromolecules (or in cord blood samples, the presence of Wharton's jelly)
may cause nonspecific aggregation that simulates agglutination if RBCs are
suspended in their own serurn.
High concentrations of A or B blood group substances in the serum
have been found, on rare occasions, to inhibit the activities of reagent
antibodies to such an extent that unexpected negative reactions are
obtained when serum- or plasma-suspended RBCs are used.
The sera of some
persons contain antibodies to the dyes used to color anti-A and
anti-B reagents. These antibodies can causefalsely positive
agglutination reactions if serum- or plasma-suspended RBCs are
used in testing. (13)
Acquired B Phenotype
Acquired B state should
be considered when the serum of a patient contains anti-B and the
patient's RBC appears to be group AB with a weak B antigen. The acquired B
phenotype arises through the modification of the A antigen by microbial
enzymes called deacetylases. The enzymes modify cellular A immunodominant
sugars (GalNAc) so they become more like the B sugar (Gal). A, RBCs are
the only group that exhibits acquired B activity in vivo.' When present in
sufficient numbers, acquired B antigens react with human anti-B in
direct agglutination tests. While many examples of RBCs with acquired B
antigens react weakly with anti-B, some examples can be found that
are agglutinated quite strongly.
To confirm that group
A1 RBCs carry the acquired B structure:
Check the patient's diagnosis. Acquired B antigens tend to be
associated with carcinoma of the colon or rectum, infection with
gram-negative organisms and intestinal obstructions.
Test the patient's serum against his or her own RBCs. The
anti-B in the patient's serum will not agglutinate his or her own
RBCs when they carry the acquired B determinant.
Test the RBCs with monoclonal anti-B. Some monoclonal
reagents, unlike human-source antibodies, do not react with the
acquired B phenotype - Such information may be carried in the
instructions that accompany the monoclonal reagent. Test the RBCs with
human anti-B serum. acidified to pH 6.0. Acidified anti-B sera
do not react with the acquired B receptor.
If the patient is a secretor, test saliva for the presence of A and
B substances. Patients whose RBCs carry acquired B structures will have A,
but not B, substance in their saliva.
Acquired A-Like Antigens
ABO discrepancies are
sometimes associated with Tn polyagglutination. (1,13) Tn activated RBCs
have glycoproteins that carry improperly formed oligosaccharides. Such
structures appear when there is a genetic dysfunction in a hematopoietic
stem cell resulting in a deficiency of a particular glycosyltransferase.
When group O ,Tn or group B,
Tn RBCs are tested, they may behave as if they have acquired an
A-like antigen reacting with human or monoclonal anti-A
reagents. The A like antigen of Tn RBCs can be differentiated from A
arising through the action of an A gene transferase if RBCs are treated
with proteolytic enzymes before testing. A-like antigens of Tn RBCs
are destroyed by enzymes.
In some cases, samples
are encountered that contain two distinct, separable populations of RBCs.
Usually a mixture occurs because group O RBCs were transfused to a group A
(or group B) patient. RBC mixtures also occur in a condition called
chimerism, resulting either from the intrauterine exchange of
erythropoietic tissue by fraternal twins or from mosaicism arising through
dispermy. Less frequently, it occurs when a patient has received a
transplant of bone marrow that is of an ABO group different from the
agglutination is characteristically seen when A3 RBCs are tested with
anti-A. If the agglutinated RBCs are removed and the remaining RBCs
again tested with anti-A, mixed-field agglutination occurs in
the residual population as well. Mixed-field agglutination may also
be seen with RBCs carrying A antigens weakened by diseases such as
leukemia or with Tn RBCs.
Antibody-Coated Red Blood Cells
RBCs from infants with
HDN, or from adults suffering from AIHA or HTRs may be heavily coated with
IgG antibody molecules. Such RBCs often agglutinate spontaneously in the
presence of high protein reagents such as anti-D. In some cases,
sensitization is such that the RBCs also agglutinate in low-protein
coated heavily with IgM cold reactive autoagglutinins will agglutinate
spontaneously in saline tests. If the RBCs are washed several times with
saline warmed to 37 C, the antibodies can be eluted from the RBC
alloantibodies, such as anti-P, or anti-M, react at room temperature
and may agglutinate the reagent RBCs used in serum grouping that have the
corresponding antigen. In general, reagent RBCs used for antibody
detection will also be agglutinated at room temperature. (Rarely, the
serum may react with an antigen on the RBCs other than A and B that is not
present on the antibody detection RBCs.) To determine the correct ABO
group of sera containing other cold-reactive alloantibodies:
Identify the alloantibody, as described in Chapter 15.
Test the reagent A, and B RBC to determine which reagent, if
either, carries the corresponding antigen.
Test the serum against examples of A, and B RBCs that lack the
corresponding antigen. For instance, if anti-M is identified, test
the serum against A,, M - and B, M RBCs to resolve the discrepancy.
If the antibody detection test is negative, repeat serum ABO tests with
several examples of A, and B RBCs. Since the antibody is directed against
an antigen of low frequency, most randomly selected A, and B RBCs will
lack the corresponding antigen.
from patients with abnormally high concentrations of serum proteins, who
have altered serum protein ratios or who have received plasma expanders of
high molecular weight can cause reagent RBCs to appear agglutinated. Some
of these samples cause rouleaux to occur. Rouleaux formation can be easily
recognized microscopically if the RBCs aggregate in what have been
described as "stacks of coins." More often, such sera cause
aggregates that appear as irregularly shaped clumps that closely resemble
antibody mediated agglutinates.
The H System
mentioned previously, the genes of the H blood groups are H and h. H leads
to the production of the H antigen that serves as the precursor molecule
on which A and B antigens are built. The amount of H antigen is, in order
of diminishing quantity, O>A2>B>A2B>A1>A1B. H like antigens
are found in nature, and persons of the rare Oh phenotype, whose RBCs lack
H, have (in addition to anti-A and anti-B) potent anti-H
in their serum that is considered to be clinically significant .(21)
Occasionally, group & A,B or (less commonly) B persons have so little
unconverted H antigen on their RBCs that they may produce anti-H. In
such situations the antibody is relatively weak and virtually always
reacts at room temperature or below. In contrast, the anti-H of Oh
persons reacts well over a wide thermal range (from 4-37 C)
with all RBCs except those of other Oh people. Oh patients must be
transfused with only Oh blood because their anti-H rapidly destroys
the H + RBCs of the other ABO groups. (28)
"Bombay" has been used for the Oh phenotype because examples of
such RBCs were first discovered in Bombay, India. The symbol Oh has been
selected to denote the phenotype bemuse results obtained in routine ABO
grouping tests mimic those of group O persons. Oh RBCs are not
agglutinated by anti-A, anti-B or anti-A,B. That a
sample is Oh (and not group O) is generally not recognized until serum
from the Oh person is tested against group 0 RBCs. Group 0 RBCs are
agglutinated by Oh sera as strongly as A and B RBCs. The Oh phenotype can
be proven if the RBCs are tested with the anti-H lectin of Ulex
europeaus. Anti-H lectin fails to agglutinate Oh RBCs, although it
agglutinates group O RBCs quite strongly. Further confirmation testing can
be performed if other examples Of Oh RBCs are available. The serum of a
suspected Oh person will be compatible only with the RBCs of other Oh,
Para-Bombay Phenotypes, & and
Ah and Bh RBCs lack
serologically detectable H antigen, but carry small amounts of A or B,
depending on the genotype of the donor. Weak reactions are obtained in
grouping tests with anti-A or anti-B.(6,11,13) The RBCs are
nonreactive with anti-H lectin or with the anti-H sera of Oh
Persons. The para-Bombay phenotype is thought to result from the
inheritance of variant H genes that produce only minute amounts of H
antigen. All of the H is converted to A or B by the products of the A and
B genes, respectively. The sera of Ah and Bh people contain anti-H
in addition to the expected anti-A or anti-B.
The Lewis Antigens
The common Lewis
antigens, Lea and Leb, are not intrinsic to RBCs, but are carried on
plasma glycosphingolipids that are adsorbed from plasma to the RBC
membranes. Their presence or absence in plasma and on RBCs is dependent,
in part, on whether a person has inherited one Le or two le genes. The Le
gene encodes a fucosyltransferase that adds Fuc in alpha(1-4)
linkage to the subterminal
of Type I oligosaccharides. (11) The resulting
structure has Lea activity. Persons who have inherited the dominant Se(H)
gene in addition to Le produce an antigen called Leb. When Leb is produced
it is adsorbed preferentially over Lea to RBC membranes. Leb is made
when Type I chains are first modified into H by the Se(H) gene
transferase. The Le gene transferase then adds Fuc to this structure to
form Leb. The le gene is an amorph. Persons who are lele produce no Lea
and no Leb antigens. Table 10-4 shows the Lewis phenotypes, together
with their frequencies in the population. RBCs that type as Le(a+b+) are
only rarely found when human antisera are used in typing. Such RBCs are
seen more frequently when more potent monoclonal anti-Lea and
anti-Leb reagents are used.
Lewis antibodies occur
almost exclusively in the sera of Le(a-b-) people, and usually
without known RBC stimulus. People whose RBC phenotype is Le(a- b+)
do not make anti-Lea because small amounts of unconverted Lea are
present in their saliva and plasma. It is unusual to find anti-Leb
in the serum of a Le(a+ b-) person. Anti-Lea and anti-Leb may
occur together in sera. They are almost always IgM and do not cross the
placenta. Because of this, and because Lewis antigens are poorly developed
at birth, the antibodies have not been implicated in HDN. Lewis antibodies
may bind complement. Fresh sera that contain anti-Lea (or
infrequently anti- Leb) may cause the in vitro hemolysis of
incompatible RBCs. In vitro hemolysis is more often seen with
enzyme-treated RBCs than with untreated RBCs.
Lewis antibodies agglutinate saline-suspended RBCs of the
appropriate phenotype. The resulting agglutinates are often fragile and
are easily dispersed if RBC buttons are not resuspended gently after
centrifugation. Agglutination sometimes is seen after incubation at 37 C,
but rarely of the strength seen in tests incubated at room temperature.
Some examples of anti-Lea, and less commonly
anti-Leb, produce positive indirect antiglobulin reactions,
providing complement is present in the reaction mixture and polyspecific
antiserum is used.
with anti-Leb activity can be divided into two categories. The most
common examples react best with RBCs of group O and A2 These have been
designated as anti-Lebh. Those that react equally well with the Leb
antigen on RBCs of all ABO phenotypes are called anti-LebL .
Anti-Lebh , but not anti-Le bL is neutralized by saliva from
group O, Le(a- b-) persons who are secretors of H substance.
Table 10-5 lists the serological behavior of the common Lewis system
additional antibodies have been given names in the Lewis system although
the determinants with which they react are not determined by Lewis genes.
Anti-Lec has been reported in one human subject as a
cold-reactive agglufinin. This
antibody agglutinated the RBCs; of Le(a-b-) people who are
sese and are therefore nonsecretors, of H substance. The antibody called
anti-Led agglutinates the RBCs of Le(a- b-) secretors.
The product defined by anti-Led has been identified as the Type I
oligosaccharide to which Fuc has been added at the H-active site.
Anti-Led should more correctly be called anti-Type I H. The
material that reacts with anti-Lec seems to be the Type 1 chain with
no added Fuc molecules. No examples of anti-Le d have been found in
humans but both anti-Lec and anti-Led have been successfully
produced in goats injected with saliva from Le(a- b-)
nonsecretors and Le(a- b-) secretors of H, respectively.
Lewis Antigens in Children
RBCs from newborn
infants usually fail to react with both human anti-Lea and
anti-Leb and, thus are considered to be Le(a- b-). Some
can be shown to carry small amounts of Lea when tested with potent
monoclonal or goat anti-Le a reagents. Reliable Lewis grouping of
young children may not be possible, as test reactions may not reflect the
correct phenotype until 6 years of age. Among children, the incidence of
Le(a+) RBCs is high and that of Le(b+) RBCs low. The phenotype Le(a+ b+)
may be observed as a transient phase in children whose phenotypes as
adults will be Le(a- b+).
RBCs are agglutinated by certain sera that agglutinate the Le(a+ b-
) and Le(a-b+), but not Le(a-b-), RBCs of adults.33 In
serological. tests, these sera appear to contain inseparable forms of
anti-Lea and Le b. They define a determinant that has been called
Lex, and which is present on the majority of cord RBCs and on the Le(a +)
or Le(b +) RBCs of adults. Many serologists, have suggested that
anti-Lex may represent a more potent or more avid form of anti-Lea.
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