Blood groups and the inherent differences in human blood from one
individual to another were first discovered by a German scientist, Karl
Landsteiner in 1900. He took samples of blood from six of his colleagues,
separated the serum and prepared saline suspensions of the red cells. When each
serum sample was mixed with each cell suspension, he noticed that agglutination
of the cells had occurred in some mixtures and not in others.
The classification of the groups was based on the realization that
agglutination has occurred because the red cells possessed an antigen and the
corresponding specific antibody was present in the sera. When no agglutination
had occurred, either the antigen or the antibody was missing from the mixture.
Landsteiner isolated and recognized two separate antigens, which he called “A”
and “B.” The antibody that reacted with the A antigen, he called “anti–A” and
the antibody that reacted with the B antigen, he called “anti –B.”
From these facts, Landsteiner recognized three separate “groups”
named according to the antigen present on the red cells. Individuals who
possessed the “A” antigen (their cells showed agglutination with anti–A) were
classified as belonging to group A. Individuals who possessed the “B” antigen
(their cells showed agglutination anti–B) were classified as belonging to group
B. Certain individuals showed no agglutination with either anti–A or anti–B,
and they were classified as belonging to group O – the symbol “O” denoting the
lack of A and B antigen in the red cells. A fourth group was discovered by
Landsteiner’s pupil, Von Decastello and Sturli in 1902. Individuals in this
group showed agglutination with both anti–A and anti–B and the group was called
“AB.”
It was discovered that individuals who possessed the A antigen on
their red cells also possessed anti–B in their serum. Individuals who possessed
neither A nor B antigen (group O) has both anti–A and anti–B in their serum and
individuals with both A and B antigens showed neither anti–A nor anti–B in their
serum.
It appears likely, however, that naturally occurring anti–A and
anti–B are, in reality, heteroagglutinins produced as an immune response to
substance which are antigenically similar to the blood group substances in
humans. It is known that bacteria contain substances very similar to human A
and B substance, and it appears evident that this case can be either infected
or inhaled.
The A and B antigens are inherited, though they may not express
themselves at full strength until about three years after birth. It is thought
that when a child who has inherited an A antigen from one or both of its
parents is bought into contact by ingestion and / or inhalation with A and B
substances in bacteria, the B substance is not recognized and is considered as
a foreign and potentially harmful substance, and an antibody – anti–B – is
formed to protect the body from the invasion of this substance. This theory is
supported by two facts:
First, newborn infants do not possess the anti–A and / or anti–B
antibodies.
Second, all persons who belong to blood group A have anti–B in
their plasma.
Individuals who belong to blood group B accept the B substance and
not the A substance and produce anti–A. Persons of blood group O accept neither
substance and produce both anti–A and anti–B and persons of blood group AB
accepts both substances and therefore produce neither anti–B nor anti–A.
****** INHERITANCE OF THE ABO GROUP ******
The A and B antigens like other blood group antigens; are the
expression of genes inherited from the previous generations. If an antigen is
demonstrated, the gene controlling it must have been inherited from or on both
of the parents, and these genes can be passed to the next generation.
ABO phenotype and genotype
Phenotype Genotype
A AA
AO
B BB
BO
AB AB
O OO
Group Antigens on red cells Antibodies in plasma
A A anti–B
B B anti–A
O none anti–A and anti–B
AB A
& B none
The genes, A, B and O are alleles, i.e., any of the three occupy
the ABO locus on each pair of chromosomes responsible for this system. If the
chromosome inherited from the father carried the gene A and the chromosome from
the mother carried the gene B, the child will be of the genotype AB and his or
her red cells will have both A and B antigens. Persons who is amorph, i.e., it
does not produce a detectable antigen as in Group O. Group O cells are
recognized by the absence of A or B antigen. When the O gene is inherited with A, only the A gene expresses itself; red
cells from an individual of the genotype AO will react on the same way as cells
from someone of the genotype AA in tests with anti–A. Similarly, we cannot
differentiate BO from BB in tests with anti–B. The symbols A and B denote
phenotypes, whereas AA, BO, etc are genotypes.
When the same gene has been inherited from each parent, for example,
OO, the individual is said to be homozygous for that gene. When different genes
are inherited, as is evident in the case of AB, the individual is heterozygous.
The genotype of someone of the phenotype A (or B) may be deduced from a study
of his family; the person is homozygous AA will pass an A gene to each of his
children while the heterozygous AO individual may pass either an A or an O gene
to his offspring. As for all chromosomes, chance alone determines which of the
pair ends up in zygote from which the new individual results.
Since each of the pair of chromosomes of an AB person carries
either an A or B gene, an AB parent cannot normally have a group O child. By
the same reasoning, someone who is group O cannot have an AB child because the
child must inherit one O gene that parent and the gene from the other parent
could not transmit both A and B.
****** THE ABO BLOOD GROUP ANTIGEN ******
The membrane of human red blood cell is crowded with
immunologically reactive molecules of antigens. Some these antigens are protein
based, such as those found in Rh, M and N systems. The ABH antigens are formed
by the attachment of certain terminal sugars in specific linkage configurations
to a precursor carbohydrate chain. For the attachment of these specific sugars
to occur, a particular enzyme called transferase is needed. The
production of the enzyme responsible for the addition of the terminal sugar
residues to the carbohydrate backbone is genetically controlled by the A, B, H
and O genes. The H and A, B, O genes are located at two different loci. The A,
B, O genes are located on chromosome 9; the chromosome location of the H gene
is unknown.
The H gene must be inherited and its genes product (enzyme) must
be present for the A, B, O genes to be expressed. Given a properly functioning
H gene, the inheritance of an A or B gene will result in the expression of A or
B antigen on the red cell surface. However, the O gene (an amorph) does not
code for the production of additional detectable component, leaving only the
product of the H genes demonstrable serologically.
The H antigen
An individual possessing an H gene (HH or Hh) will produce an
enzyme (tranferase), alpha–L–fucosyltransferase, which causes H
antigen to be produced by attaching an L–fucose to the D–galactose portion of
the precursor chain.
The resulting antigen can be detected with anti–H serum or lectin–H.
Those individuals who are homozygous “hh” lack the H gene, thus the attachment
of ABO specific sugars to H substance are prevented. Consequently, regardless
of the presence of an A or B gene, the A or B antigens will not be expressed
and the individual will be typed as group O. These unusual cell types are classified
as “Bombay” and are additionally characterized by the presence of potent anti–H
in the autologous.
The A antigen
The presence of the “A gene” codes for the production of A–transferase.
This enzyme adds N–acetyl–D–galactosamine to the terminal D–galactose of H,
thus producing the A antigen.
Approximately 75–80% of group A individual (called A1)
have little or no “H antigen” serologically demonstrable on the surface of their
red cells. The remaining 20–25% of “group A” individuals possess a weaker variant
(A2, A3, Am, etc.) of the “A antigen” and have
significant levels of red cell H antigen. The differences in serologic
reactions encountered in testing for the presence of the “A antigen” are the
result of quantitative and / or qualitative variations. This difference in “A
antigen” maybe due to slight physical differences in the biologic efficiency of
the alpha–N–acetyl–galactosaminyl transferases.
The B antigen
The presence of the “B gene” codes for the production of
B–transferase to add B–galactose to the terminal D–galactose of H, producing
the B antigen.
The “Bombay” phenotype (Oh)
The first example of what has come to be known as the “Bombay”
phenotype was reported in 1952 by Bhende and his associates. Although the
condition seems to be common in the Marathi–speaking people of India, about 30
such bloods have now been recognized in various parts of the world.
The red cells of a person with the “Bombay” phenotype are devoid
of antigens of the ABO system; in routine tests, the cells are not agglutinated
by anti–A, anti–B or anti–H. The lack of reaction with anti–A and anti–B
suggests that red cells are from the “group O” individual, however, group O cells
react it strongly with anti–H. Serum samples from “Bombay” individuals contain
anti–A, anti–B and anti–H. The presence of anti–H in the individual’s serum is
a further indication that his or her red cells are not “group O.” Because of
the multiplicity of antibodies in the serum, the “Bombay” transfusion recipient
is incompatible with donors of groups A, B, AB and O. Only the blood of another
“Bombay” person could be compatible.
The “Bombay” phenotype is due to the absence of the common gene H,
or, to put it another way, the presence of h in the homozygous state. H is
required for the formation of “H antigen,” the substrate to which A and/or B
genes of “Bombay” persons appear to be normal since they express themselves by
producing antigen in other members of the same family who possess at least on H
genes. Hh children, the mating of hh x HH (one parent “Bombay,” the other
normal) have normal A, B and H red cell antigens. Presumably, the A and B
sugars are present in “Bombay” but cannot be attached to anything but H
substrate, thus, no A, B or H antigen is found in the absence of an H gene.
The genotype hh gene usually occurs in the offspring of CONSAN–GUINEOUS
MARRIAGE. Frequently, the parents are cousins and both are of the genotype Hh,
however, a recent report by Units and his associates describes a family in
which one parent was Hg and the other hh.
****** THE ABO BLOOD GROUP ANTIBODIES ******
Persons who are exposed to an antigen that they lack may respond
by producing an antibody specific for that antigen. Since most blood group
antigens are restricted to red cells, production of a blood group antibody usually
requires the introduction of foreign red cells by transfusion or pregnancy.
However, the A and B antigens are among those whose structure closely resembles
antigens of bacteria and plants to which we are constantly exposed. As a result
of this exposure, virtually everyone over the age of six months who lacks
either or both of the antigens A and B found in the antibody. The regularly
occurring anti–A and /or anti–B found in the serum of all but the people of
group AB provides a convenient source of test reagents and also affords an
ideal check on the red cells group.
Time of appearance
Antibody production does not normally begin until after birth.
Newborns have their mother’s IgG antibodies but these are passively received,
not actively produced. Anti–A and anti–B production begins in the first few
months of life with titers rising for the first five or six years and then
remaining functionally the same until late in adult life. In very old people,
levels of anti–A and anti–B are significantly lower than in young adults.
Antibody behavior
Agglutination is the most conspicuous serologic effect of anti–A and
anti–B. Other effects can and do occur under appropriate circumstances.
Hemolysis, an important in vivo effect, sometimes occur in vitro condition and
should be sought in observing every serum test. ABH antibodies sometimes coat
cells without causing agglutination. When coating and agglutinating antibodies
of the same specificity are present in serum, only agglutination is apparent,
unless the agglutinating antibodies are neutralized or inactivated. They are
clinically important in hemolytic disease of the newborn since like all IgG
antibodies, they readily cross the placenta.
Antibody characteristics
1.
Agglutination of saline suspended erythrocytes
2.
Reactivity at room temperature
3.
Production of hemolysis in vivo and in vitro.
Both IgM and IgG anti–A and anti–B are capable of binding complement. All
examples of IgG anti–A and about 90% of IgM anti–A have been demonstrated to be
hemolytic.
4.
Inactivation of IgM anti–A and anti–B with
2–mercaptoethanol (2–ME)
Anti–A,B serum (Group O serum)
The commercially available reagent used to group red cells are
prepared from the serum of group A persons who have produced anti–B and group B
persons who have produced anti–A. The serum of selected group O persons is used
to prepare an additional reagent anti–A,B which has the ability to detect weak
forms of tests with anti–A and anti–B. Anti–A,B serum will agglutinate A, B and
AB cells but not cells of group O.
Anti – H
1.
Human anti–H are of two types
a.
Anti–H produced as cold agglutinins. This
reacts at low temperature and is a natural antibody. This antibody reacts most
in strongly with cells of group O, group A2 and weaker subgroups and
reacts hardly at all with group A1 and A1B cells.
b.
Anti–H formed by Bombay individual. These are
active over a wide range of temperature and may lyse red cells.
2.
Lectin anti–H – an extract from the seeds of
the plant Ulex europaeus, gives reactions that closely parallel
of human anti–H.
Natural antibodies
These are non–red cell stimulated IgM antibodies formed early in
life, probably due to exposure to plant or bacterial antigen. Naturally
occurring anti–A and anti–B react best at temperatures below 37oC
when tested against saline suspended red cells.
Immune antibodies
Agglutinating anti–A and anti–B develops so regularly after
environmental exposure that they are considered naturally occurring, i.e., no
recognizable immunizing event leads to their appearance. A person exposed to a
specific immunizing event may produce antibodies of the same specificity but
different biologic behavior.
Immunizing
events include:
1.
Pregnancy with an ABO–incompatible fetus.
2.
Transfusion of incompatible red blood cells
or of plasma containing blood group substances.
3.
Inoculation with viral or bacterial products
containing blood group active materials.
4.
Injection of purified blood group substances.
After immunization, the subjects’
antibody may:
1.
Increase in titer or avidity.
2.
Develop powerful hemolyzing properties.
3.
Become more difficult to neutralize with
soluble blood group substances.
4.
Become more active at 37oC.
These changes are more common in group O subjects but may occur in
group A or group B persons as well. Although the distinction is not always
complete, naturally occurring antibodies tend to be IgM and “immune” activity
is more often IgG.
Weak and Missing Antibody reactions:
Missing or weak isoagglutinins result from depressed or absent
antibody production. This may be caused by the following:
1.
Age. Newborn and young infants and
the elderly may exhibit weak or missing isoantibodies. Detectable antibodies in
newborn or young infants are acquired in utero from the mother.
2.
Hypogammaglobulinemia. Decreases in the gamma globulin
fraction of plasma proteins can lead to weak or missing antibodies. Conditions
in which hypogammaglobulinemia may be demonstrated include the use of
immunosuppessive drugs, lymphomas, leukemias, immunodeficiency disorders and
following bone marrow transfusion.
3.
Agammaglobulinemia. This disorder maybe acquired or
congenital. Disorders such as Burton’s agammaglobulinemia, immune deficiency
disorders and the effects of physical agents such as radiation exposure and
cytotoxic drugs can all contribute to this condition.
4.
Chimerism. This is the production of two
cell populations in one individual throughout his lifetime. The two cell
populations are both recognized as self; consequently, these individuals do not
make anti–A or anti–B and no detectable isoagglutinins are present in the
serum. Chimerism in a person who has no twin may be caused by dispermy (two
sperm fertilizing one egg) and indicates mosaicism.
Artificial or transient chimeras
can be detected when group O erythrocytes are transferred to group A or B
patient after bone marrow transplant, exchange transfusion or fetal–maternal
bleeding.
Lectins / Phytohemagglutinins
Phytohemagglutinins are plant agglutinins; they are extracts of
beans which contain substances which react in a more or less specific fashion
with human red cells.
Examples: Anti – A1 Dolichos biflorus
Anti
– B Sophora japonica
Anti
– H Ulex europaeus
Anti
– M Ibiris amar
Anti
– N Vicia graminea
Anti
– T peanuts
Prolectins
(derived from snails)
Anti
– A Helix pomatia
Anti
– A1 Euhadra peliomphala
Bradybaena
fracticum
Anti
– B Salmo irideus
Anti
– H Eel
************ SUBGROUPS
************
Some blood specimens of types A and B are consistently less
agglutinable than others. Group A red cells can be subdivided into A1,
A intermediate, A2, A3 and Ao (Ax).
Similar distinctions can be made among group AB red cells. The difference
between one subgroup and another is at least partly quantitative; A1
cells have the greatest amount of A antigen while Ao cells have the
least. Forms of “A” weaker than “Ao” exist although they are rare.
Qualitative difference may be due to unclassified form. Subgroups of B are
rare; they are usually recognized by variations in the strength of the reaction
with anti–B and no reagent is available to distinguish among them. Subgroups of
B are B1, Bs, Bo, Bx and Bw.
Subgroup identification is important to avoid misinterpretation or
misidentification of the blood group of an individual as group O. Misidentification
of the blood group may lead to transfusion reactions.
Subgroups maybe identified by absorption with specific antisera
followed by elution of antibodies from red cells and identification of the
antibody in the eluate.
Absorption – removal of antibodies from serum by the
addition of red cells that possess the corresponding antigen.
Elution – the removal of antibody from red cell
surface (Elution technique is found in Lecture #9).
Eluate – a fluid medium containing antibodies which have been
deliberately removed from the red cell.
Reagents used to identify subgroups
1.
Anti–A – comes from serum of group B
individual; reacts with cells and subgroups of A.
2.
Absorbed anti–A serum (Anti–A1) –
absorbing group B serum with A2 cells leaves anti–A1
only.
3.
Anti–A,B serum (Group O serum) – comes from
serum of group O individuals, agglutinates Ao, Ax and Am
besides the regular A and B agglutinogen.
4.
Anti–A1 lectin – prepared from the
seed of Dolichos biflorus; agglutinates A1 and A1B
cells.
5.
Anti–H lectin – comes from the seeds of Ulex
europaeus, reacts less strongly with A2 and A3
cells and less regularly or weakly with A1 cells.
****** BLOOD GROUP SPECIFIC SUBSTANCES ******
(WITEBSKY SUBSTANCES)
Blood group specific substances are soluble antigens. When a
specific substance is added to its corresponding antibody, the antibody
combines with the soluble antigen and is no longer able to react with the same
antigen on red cells. The interaction of antibody with soluble antigen is
recognized by inhibition or neutralization test
Commercially available blood group specific substances A and B are
extracts of hog and horses stomachs respectively. The “B substance” usually
contains some “A substance” as well.
These extracts are used in tests to determine the presence of
immune anti–A and anti–B in human serum. While the regularly occurring
isoagglutinins are neutralized by the addition of small amounts of specific
substances to the serum, immune forms of the antibodies remain active unless
large quantities of substance are added. Following the addition of small
amounts of specific substances, the activity of the antibodies is determined by
testing the serum with A and B cells.
****** A, B and H SUBSTANCES IN SALIVA ******
(SECRETORS AND NON–SECRETORS)
Approximately 80% of the Caucasian population secretes soluble
antigenic substances in saliva which have the same specificity as the antigens
on the red cells. The term “secretor” refers to an individual who secrete A, B
and H substances of other blood groups, also found in saliva, and are not taken
into account.
The appearance of ABH antigen in saliva is regulated (modified) by
a set of secretor (Se or se) genes which are inherited independently of ABO and
H genes. The relevant gene is called Se and its allele se.
individuals who are homozygous or heterozygous for the Se gene are secretors
but individuals who lack the Se gene are therefore homozygous for the amorphic
allele, se, are non–secretors.
All secretors secrete H substance, regardless of their ABO groups.
Thus the saliva of a “group O” secretor contains H substance; the saliva of “group
A” secretor contains A and H substances; the saliva of “group B” secretor
contains B and H substances, and the saliva of a “group AB” secretor contains
A, B and H substances.
Non–secretors do not secrete H, A or B substances regardless of
ABO group since Se is a regulator gene which permits H to be expressed in
secretory tissue. Since H appears to be a necessary precursor of A and B,
non–secretor cannot secrete A or B. A person is a non– secretor if he does not
inherit the Se gene.
A, B and H substances have been found in almost all body fluids
including seminal fluid, urine, sweat, tears, digestive juices, bile and milk.
****** ABO GROUPING
******
ABO grouping is the most important laboratory test performed on
potential transfusion recipients and blood donors. The critical nature of ABO
grouping stems from two characteristics of the system.
1.
Antibodies of the ABO system are present in
the serum of almost every person who does not have the corresponding antigen.
2.
The alloagglutinins of the ABO system fix
complement and are capable of causing intravascular hemolysis of incompatible
red cells.
Two types of ABO grouping
1.
Cell grouping (forward or direct typing) –
determination of red cell agglutinogens by testing red cells with anti–sera of
known specificity.
2.
Serum grouping (reverse or indirect typing) –
determination of the isoantibodies in the serum using red cells of known
specificity.
Expected reactions in ABO grouping
Group AB
Group AB is called the “universal recipient,” because the
erythrocytes of the other three groups are not agglutinated by the serum of the
group AB (i.e., no agglutinins are present in the serum of this group.
Group A
The serum of this group agglutinates the erythrocytes of groups A
and AB only, whereas the erythrocytes of this group are agglutinated by the
serum of either group B or O.
Group B
The serum of this group agglutinates the erythrocytes of groups A
and AB only, whereas the erythrocytes of this group are agglutinated by the
serum of either group A or O. Human red cells of blood group B react uniformly
with anti–B serum.
Group O
The erythrocytes of this group are not agglutinated by the sera of
the other three groups. Likewise, the erythrocytes of the other three groups
are agglutinated by the serum of group O blood. In an emergency, group O blood
may be given to patients of group A, B or AB provided that the isohemagglutinin
titer (the naturally occurring anti–A and anti–B) of the group O donor is 1:50
or less or provided the titer is reduced by packing the red cells and
transfusing these packed cells and not the plasma. The tissue of certain
animals contains substances having the property of neutralizing these
isohemagglutinins. Blood group substance A is derived from gastric mucosa of
hog and blood group substance B is from gastric mucosa of the horse. Sterile solutions
of both these substances can be used to reduce the naturally occurring
antibodies but the practice of adding them to units of blood is strongly
condemned and is both dangerous and useless practice, as these substances do
not neutralize the immune forms of anti–A or anti–B.
Discrepancies between cell and serum results
When the results of cells and serum testing for ABO do not agree,
the discrepancy must be investigated. If the blood from a donor, the unit
cannot be used for transfusion until the discrepancy is resolved. When the
blood is from a patient, it sometimes becomes necessary to transfuse compatible
blood before investigations are complete. An adequate sample of pre–transfusion
blood should be obtained before donor blood is given.
Discrepancies maybe result of:
1.
Improper technique
a.
Dirty glassware may cause false positives.
b.
Improper concentration of cells to serum may
cause false positive or false negative.
c.
Failure to identify hemolysis as a positive
reaction causes false negative.
d.
Over centrifugation or under centrifugation
causes a false positive or false negative result.
e.
Carelessness on reading or failure to use
optical aid may cause a false negative.
f.
Warming cell–serum mixture may cause a false
negative.
g.
Contamination or inactivation of reagent
serums may cause a false negative or, occasionally, false positive.
h.
Incorrect identification of specimen,
materials or incorrect recording of results or interpretation causes false
positives, false negatives and total disaster.
2.
Of intrinsic properties of the
red blood cells
a.
The cells appear to be agglutinated because of
something in the patient’s serum (Wharton’s jelly or serum proteins causing
rouleaux) remains in the cell suspension tested.
b.
The patient may have antibody–coated cells, which
agglutinate in a high protein medium.
c.
The patient may have received transfused
cells and the sample is a mixture of cell types.
d.
The A or B antigen may be weakly expressed
because of unusual genotype.
e.
The A or B antigen may be weakened by the
effects of leukemia or non– hemolytic malignant conditions.
f.
The cells may have genetic or acquired
surface abnormalities that render them polyagglutinable.
g.
There may be acquired “B–like” activity,
usually resulting from action of gram– negative organisms.
3.
Of intrinsic properties of the
serum
a.
High concentrations of fibrinogen or of
abnormal proteins or altered proportions of globulins may cause rouleaux formation
which resembles agglutination.
b.
There maybe an unexpected antibody reacting
with A, B or H antigens. The most common are anti–A1 in A2B
serums and anti–H in A1 or A1B serums.
c.
There may be an unexpected antibody in some
other blood group system, reacting with antigen on the A or B cells used for
serum testing. Anti–I is probably the most common troublemaker.
d.
The patient may have been given dextran,
intravenously injected contrast materials or drugs that cause cellular aggregation
that resembles agglutination.
e.
The patient may have an immunodeficiency
disease and lack expected antibodies because of low overall immunoglobulin
levels.
f.
The patient may be infant, who has not begun
producing his own antibodies or who has antibodies passively received from
another.
g.
The patient may be an elderly person whose
antibody levels have declined severely.
h.
The patient may have antibodies against
elements of the preservatives, suspending medium or reagent solutions (like
acriflavin yellow) used in testing.
Resolving discrepancies
Many of the problems can be resolve by repeating the test, washing
the cells thoroughly before testing them. Obtaining a new sample of blood from
the subject corrects difficulties introduced by contaminated specimens.
The most frequent cause for persisting problems is the presence of
rouleaux–producing factors in serum: presence of unexpected antibodies;
presence of anti – I, which reacts with nearly all cells, including the patient’s
own cell; and the presence of antibodies coating the cells (positive
antiglobulin test). In handling persistent problem, be sure to:
1.
Look at results of an antibody–screening test.
If the test is positive, identify the antibody. For serum–testing, use A and B
cells that lack the antigen involved, if these can be obtained.
2.
Determine subject’s age, diagnosis, previous
medications or transfusion and serum protein findings.
3.
Perform antiglobulin test on patient’s cells.
4.
Perform an autologous control test, using
patient’s serum and suspension of the patient’s cells for autoantibody.
****** THE A B H ANTIGENS IN DISEASE ******
Blood group antigens are normally stable throughout life; however,
in some disease states the antigens appear to be alerted. Antigens previously
demonstrated on the red cells of a patient may no longer be detectable or the
cells may acquire a pseudoantigen.
The cells of some “group A” patients with leukemia show variable
reactions with anti–A during course of the disease. When the cells are not
agglutinable anti–A, they will absorb the antibody and can be demonstrated in
the saliva if the patient is a secretor. Weakening or loss of “A antigen” from
the cells does not lead to the production of anti–A so these patients will
reverse group as expected of group A individuals. Sometimes, however, the anti–B
in their serum is weaker than that of a healthy “group A” person.
The “acquired B” phenomenon affects the red cells of some “group A”
individuals with intestinal obstruction, carcinoma of the colon or rectum and
other disorders of the lower intestine. Increase permeability of the intestinal
wall allows the passage of bacterial polysaccharide (from E.coli) into the circulation.
The patient’s group A cells absorb the B– like polysaccharide and the cells
then react with anti–B as well as with anti–A.
Patients with carcinoma of the stomach or pancreas are sometimes difficult
to group correctly, not because their red cell antigens have changed but
because their serum contains excessive amount of soluble antigen (substance)
which neutralize the antiserum employed in the test. The cells can be grouped
accurately once they have been washed free of the patient’s serum.
****** THE A B H SECRETOR STATUS ******
Principle
Certain blood group substances, namely A,B,H and Lea
occur in soluble form in secretions such as saliva in a large proportion of
individuals, referred to as secretors. These water–soluble substances are
readily detected in small quantities because they neutralize or inhibit the
capacity of their corresponding antibodies to agglutinate erythrocytes possessing
the corresponding antigen. These reactions, termed hemagglutination inhibition,
provide a means of assaying the relative strength or potency of the water–soluble
blood group substances. Identifying the presence of ABH or Lea substance can be
important in blood type problem solving.
Specimen
1.
Have the patient chew a piece of paraffin wax
(gum or anything else that contains sugar or protein cannot be used) to
stimulate salivation.
2.
Collect about 2–3 ml of saliva in a test
tube.
3.
Place the stoppered tube of saliva in a
boiling water bath for 10 minutes. Heating the specimen inactivates enzymes
that might destroy blood group substances.
4.
Centrifuge at 3,400 rpm for 10 minutes.
5.
Transfer the clear or slightly opalescent
supernatant to a clean tube. Discard any opaque or semi–solid material.
6.
The supernatant fluid should be refrigerated
until the time of testing. If testing will not be done on the day of collection
and processing, it should be frozen. Frozen samples retain their ability for
several years. Do not freeze an aliquot once it has been defrosted.
Reagents
Commercial lectin
anti–H
2–5% washed
erythrocytes, Group O
Test tubes
Pipettes
0.9% NSS
Procedure
1.
Preliminary Antisera Dilution
a.
Prepare serial dilutions, e.g. 1:2, 1:4 of
lectin anti – H.
b.
Label a clean 12 x75 test tube for each
dilution and add 1 drop of the dilution.
c.
Add one drop of the 2 – 5% saline suspension
of group O red blood cells to eah of the tubes.
d.
Centrifuge for 15 seconds at 3,400 rpm and
observe macroscopically for agglutintation.
e.
Select the highest dilution of antiserum that
produces +2 macroscopic agglutination as the working antiserum (antibody
dilution). Record results.
f.
Prepare a sufficient quantity of this
dilution to complete the test procedure.
2.
Detection of secretion
a.
Label four 12 x 75 mm test tubes: patient,
reagent control, Se and se.
b.
Add one drop of the patient’s supernatant
saliva to the patient tube. Add 1 drop of saline to the reagent control tube
and 1 drop of known Se and se controls to their respective tubes.
c.
Add one drop of working antibody dilution to
all tubes
d.
Incubate all the tubes at room temperature
for 10 minutes.
e.
Add one drop of the 2 – 5% red blood cell
saline suspension of group O cells to each tube.
f.
Incubate all the tubes at room temperature
for 30 – 60 minutes.
g.
Centrifuge for 15 seconds at 3,400 rpm.
h.
Observe for macroscopic agglutination and
record results.
Interpretation
The reagent control should demonstrate +2 agglutination. The Se
control should demonstrate no agglutination and the sese control should be
negative (no agglutination). A non–secretor shows agglutination of red cells by
antiserum–saliva mixture. A secretor shows no agglutination of red blood cells
by antiserum–saliva mixture.
Note: To detect or measure
salivary A or b substance, use the same procedure with diluted
anti–A or anti–B.
PROCEDURE FOR DETECTION OF LEWIS SECRETION
CONTROLS: Use
saliva from a person whose red cells are Lea positive and from one
who
Is Lewis–negative
1.
Prepare working Lea antiserum in the same
manner as lectin anti –H.
2.
Label four 12 x 75 mm test tubes: patient,
reagent control, Lewis–positive and Lewis –negative.
3.
Add one drop of processed saliva to the appropriate
tube and a drop of saline to the reagent tube.
4.
Add one drop of working diluted antiserum to
all tubes.
5.
Incubate for 10 minutes at room temperature.
6.
Add 1 drop of 2 – 5% saline suspension of
washed Lea red blood cells to each tube.
7.
Incubate for 30–60 minutes at room
temperature.
8.
Centrifuge at 3,400 rpm for 15 seconds.
9.
Observe macroscopically for agglutination.
10. Record
results.
Interpretation:
A non–secretor of Lewis substance demonstrates agglutination; a
secretor has no agglutination of the Lea positive indicator cells. The
reagent control should exhibit 2+ agglutination.
A Lewis positive person who is a secretor of ABH can be assumed to
have Leb as well as Lea in the saliva. A Le(a+) person
who is sese and does not secrete ABH substance will only have Lea in
saliva.
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