In 1939, Levine and Stetson published their findings on the serum
of a group O patient who had suffered a transfusion reaction after receiving
the blood of her group O husband. The patient had not been transfused previously;
she had just completed her second pregnancy with the delivery of a macerated
fetus. Serum drawn from the patient after the transfusion reaction was found to
agglutinate the husband’s cells as well as 80 of 104 red cell samples. Levine
& Stetson suggested that the woman had produced an antibody specific for a
fetal antigen genetically transmitted from the father and this antibody was
responsible for the transfusion reaction. No name was given to the antibody.
It received its name in 1940, when Landsteiner and Wiener
immunized rabbits with red blood cells from Rhesus monkeys and found that the
rabbit anti–Rhesus antibody agglutinated approximately 85% of human red blood
cells tested. They gave the name “Rh” to this determinant present on all Rhesus
monkey cells and apparently present in 85% of human red blood cells. Levine and
his co–workers found several other post–partum women with similar antibodies,
at least one of which gave reactions parallel to rabbit anti– Rh and Wiener and
Peters observed human examples of
anti–Rh in Rh negative patients who had received ABO–compatible, Rh–positive
transfusion.
Rh–positive and Rh–negative refer to the presence or absence of a
single red blood cell antigen, respectively.
Now called Rho(D), this antigen is, after A and B, the
most important antigen in transfusion practice. Unlike the situation with ABO,
persons whose cells lack the antigen do not routinely have the antibody in
their serum. The antibody almost always results from exposure, either through
transfusion or pregnancy, to immunizing red blood cells containing the antigen
and such exposure elicits antibody production in a high proportion of
Rh–negative subjects. As transfusion became more frequent in 1940’s, there were
increasing opportunities for immunization to occur and for antibody, once
developed, to become more apparent.
The immongenicity of Rho(D), that is, its likelihood of
provoking an antibody if transfused into a negative patient, is greater than
that of virtually all other antigen studied. Four additional antigens have been
recognized as belonging to what we now call the Rh system. They are: rh’ (C),
rh’’ (E), hr’ (c), hr’’ (e). The association of these factors suggests that
immunologic activity of Rh arises from surface materials with several different
determinant areas.
Nomenclatures
1.
Fisher–Race Nomenclature (CDE terminology)
Distinguished genes from the
observed antigenic specificities asserting that the immediate gene product is a
single entity called an agglutinogen, which is, in turn, characterized by
various serologic specifications. Wiener proposed that one gene at a single
locus on each chromosome of the pair controls the entire Rh system. The two genes
may be alike (homozygous) or different each other (heterozygous). Multiple
alleles of this gene exist: the eight major alleles are called Ro, R1,
R2, r, r’ and ry. Each gene produces an antigen on the red cell
called an agglutinogen and each agglutinogen can be identified by its parts or
factors that react with specific antibodies. Example, the gene R1
has been inherited on one chromosoms and gene r at the same locus on the other
chromosome. The gene R1 determines the agglutinogen Ra1
on the red cell and this agglutinogen is made up of at least three factors –
Rho, rh’ and hr’’. The gene r determines the agglutinogen rh on the red cell
distinguished by its factors hr’ and hr’’.
2.
Rosenfield Nomenclature
Both the Fisher–Race and Wiener
nomenclatures are based on genetic concepts of theories of inheritance.
Rosenfield, et.al, proposed a notation in which each of the various Rh antisera
is assigned a number, somewhat arbitrarily. This system permits an unbiased
phenotype determination based solely upon the results observed with the
antisera employed.
Comparison of terms in three nomenclatures
·
D (Hro) is a theoretical factor,
not yet discovered
Interpretation of Wiener and Fisher–Race systems can be
facilitated by the use of the following rules:
1.
The use of an upper case R denotes the
presence of Rho(D), while lower case r reflects its absence.
2.
The superscript ‘(prime) signifies the determinant
rh’ (C) while “(double prime) signifies the
determinant rh’’ (E).
3.
Usually, when rh’ (C) is present, hr’’ (e) is
also present and rh’’ is usually accompanied by hr’ (c).
Terminologies to remember:
1.
Antithetical – refers to two antigen controlled
by a pair of allelic genes.
2.
cis position effect - when an Rh gene or on one chromosome affects
the action of another Rh gene on the same chromosome (in terms of increase or
decrease antigen production)
3.
Compound antigen – the term used to express
the idea that certain combination of antigens demonstrate a combined effect
(e.g., ce or f antigen).
4.
Dosage effect – a variation in strength of
agglutination between homozygous and heterozygous erythrocytes. The presence of
a homozygous genotype can express itself with more antigen than the
heterozygous genotype and can produce a stronger degree of agglutination. It is
observed often with antibodies having specificities for the E, c or e antigen.
5.
Factors – agglutinogen with individual
serologic specificities.
6.
Haplotype – gene complex of an individual or
population.
7.
Rh mod – refers to the complete type of Rh
gene expression.
8.
Rh null – refers to the absence of Rh antigen
on erythrocytes membrane. Individuals exhibit stomatocytosis (cup–shaped red
cells).
9.
Trans position effect – when an Rh gene on
one of the chromosome of a homologous pair affects the action of an Rh gene on
the other homolog.
Phenotype and genotype
In clinical practice, only five reagent antiserum are readily
available. For most pre–transfusion studies, test are performed only for Rho(D)
while the other antiserums are used principally in family studies or
investigations of commonly encountered antibodies. The assortment of antigens
detectable on an individual’s cells is his phenotype. Since any individual
antigen may be part of several different genetic packages, it is no always
possible to deduce which combination of genes has produced a given phenotype.
This is important in population studies, in studies to assign parentage and in
evaluating hemolytic disease of the newborn resulting from anti–Rho
(D).
Rh phenotypes based on reactions of antisera
with erythrocytes
Selected Rh Genotypes
When D is present, it is impossible to determine (except sometimes
by a family studies) if the gene d is also present, since anti–d had never been
produced. In genotyping, therefore, its presence must be postulated using
information gained from test for the antigens C, E, D, c and e based on
probability as defined by large numbers of family studies. By comparing test
results with charts showing the incidence of the various gene complex, the
probable genotype is determined. It should be realized, however, that such
determinations are only presumptive.
****** ANTIGENS OF THE Rh SYSTEM ******
The Rh antigens are definite molecular configurations repeated in
many places on the red cell surface. Its presence or absence and that of other
antigens on the red cell are determined by gene.
The D variants
Cells of person which react weakly or slowly with anti–Rho(D)
reagents are called D variants or Du.
Du positive cells are not directly agglutinated by anti–Rho(D)
but do not react when the antiglobulin test is applied to cells.
Classification of D positive persons
This was devised in 1962 and was based on the reactions of
erythrocytes with anti–D and the specificity of anti–D produced. The six
categories are referred to as I, II, III, IV, V and VI.
I.
The anti–D produced by persons in this
category is always very weak. Erythrocytes from individuals in this category
react with all anti–D sera, except their own.
II.
Rare cases have been reported in this
category.
III.
The serum used to describe this category is
the original anti–Rhd. Individuals in this category are classified
as Rhd; most are Black; many are cDe, VS+, V–.
IV.
Some examples of anti–D sera show Go (a+)
category IV persons to have elevated D.
Black category IV members are Go (a+); White category IV members are Go
(a–).
V.
The original case in this category formed
anti–RHc. The erythrocytes of all members react with ant–Dw.
The Dw gene segregates with the unusual D gene. This category had
both Black and White members.
VI.
Only a very small proportion of anti–D
antisera react with the erythrocytes of individuals belonging to category VI.
These red cells are often called RhB (or DB); however,
the classification is a misnomer because RhB has been shown to be
normal component of the D antigen. All the members in this category are White.
Three different ways by which Du
may arise
1.
Suppression of D antigen expression because
of genetic interaction. Example is the expression of C gene over the D gene on
another chromosome due to trans position.
2.
Inheritance of a gene that codes for less D
antigen, weak D.
3.
Absence of a portion of portions of the total
material that comprises the D antigen, the D mosaic.
The D Mosaic
The concept of the D mosaic was advanced to explain the fact that
some people with D positive erythrocytes produced anti–D that was non–reactive with
their own cells. Wiener originally proposed that the D antigen on normal D
positive erythrocytes included all the components of the mosaic: A, B, C, D.
Two types of Du
1. Hereditary Du – if the parents are high grade
Du, any children who inherit Du are also high grade Du.
The antigen Du is not detectable when D is present as in the
genotype D/Du. There is no anti–Du; Rh negative persons stimulated
by Du cells may produce anti–D but they do not make anti–Du.
2. Gene interaction Du – there is red cells that appear
to be high grade Du but the trait is not inherited, hence it cannot
be considered the product of another allele of D. The genotype appears to be
usually DuCe/dCe. The C gene in trans position suppresses D on the
red cells. This is an example of gene interacting with another on the partner
chromosome to produce an effect not evident when the genes act separately as in
the next generation. When DCE is freed of the influence of dCe, there is no
longer any suppression of D.
I.
DuCe/dCe ------------- DcE / dce
II.
DCE --------------------- dCe / dce
Two grades of Du
1.
Low grade Du – detectable only by the
indirect antiglobulin test or by sensitive enzyme test.
2.
High grade Du – distinguishable from ordinary
D only by the fact that agglutination is produced by a proportion of rapid
agglutinating anti–D sera. The reaction is slow or weak.
Significance of Du
1.
Originally, a sample was classified as Du
if it reacted with anti–D antiserum but the indirect antiglobulin test only.
Distinguishing Du nowadays is complicated by the fact that many
modern slide test, “rapid tube” anti–D reagents will react even with low–grade
Du. The reaction is weaker by the slid test than with normal D+
cells.
2.
Persons who are Du+ can be transfused with D+
or D– blood; immunization is unlikely to occur if D+ blood is used. Donations
from D+ individuals are regarded as D+ and should not be transfused to D–
recipients.
3.
A Du infant can suffer from
hemolytic disease of the newborn if its mother’s serum contains anti–D;
however, immunization by the Du infant is unlikely, as Du
itself is a poor antigen.
4.
Du is relatively uncommon in
Caucasian, although it occurs commonly as cDue in Negroes.
5.
There is no specific anti–Du
serum, although Du subjects occasionally form anti–D. This is
probably because of the mosaic structure of the D antigen which is thought to
consist of four fragments, named A, E, C and D. When a fragment is missing, as
in the case with some Du individuals, anti–D may be produced against the
missing fragment, yet al of the reactions expected of ordinary anti–D is given.
Methods for the detection of Du
Samples giving negative reactions with potent anti–D must be
tested for Du. The indirect antiglobulin technique is used with
slide test (mainly IgG) anti–D especially designed for this purpose. At the
same time, a direct antiglobulin test should be performed on the cells of the
patient under test, since the presence of a D variant cannot be presumed unless
these cells give a negative direct antiglobulin test.
Generally, a sample giving a negative reaction with saline anti–D
and a positive reaction with IgG anti–D by the indirect antiglobulin test is
designed as Du, provided the auto control (direct antiglobulin test)
is negative.
Other variants
Antigen C and c may sometimes be replaed by an antigen controlled
by an alternative allelic gene. These products of alleles at the Ce locus are Cw
and Cx. Antigens Cu and Rh26 (c–like) are
weakened forms of C and c is not alternative antigens. These may result from a
normal C or c gene that is affected in its antigen production by the presence
of an independently inherited suppressor gene or modifier gene.
Like C and c, E and e can also be replaced by one of their
alternative antigens – Ew, Et, es (VS), etc.
or by a weakened form of e or E, like Eu or ei.
The production of antigen ce (f) like that of CE, Ce and cE is
thought to be controlled by the presence of two Rh genes on the same complex.
For example ce (f) is produced by rh (cde), because the c and e genes are on
the same chromosome (cis position). I, the genotype Dce / DcE, both c and e are
produced, but the blood is ce (f) – negative, since the c and e antigens are
produced by genes on different chromosome (in trans position).
The antigens G (CD) was first reported by Allen and Tippett in
1958, who described a sample of red cells which reacted with anti–CD but not
with specific anti–C or anti–D. it was also discovered by the same workers that
all red cells which have the C and D antigens also have G. most sera which
appear to contain anti–C and anti–D in fact contain either anti–D+, anti–G, or
anti–C+, anti–D, anti–G.
The LW antigen
The LW (LWa) antigen was named in honor of Landsteiner
or Wiener. The amount of LW antigen demonstrated on a person’s erythrocytes is
related to the presence or absence of D antigen.
The classification LW1 and LW2 describe the
strong LW reactivity of D+ erythrocytes and the weaker LW reactivity
of adult D–erythrocytes.
The category LW3 represents the phenotype of
individuals who produce anti–LW but have non–reactive erythrocytes with the LW
antibody.
The classification LW4 (LW a–b–) has been applied to
the extremely rare case of having LW– erythrocytes with serum containing
a potent form of anti–LW that agglutinated LW3 erythrocytes as well
as LW1 and LW2 erythrocytes. LW is a very high incidence
antigen. All erythrocytes of the Rh null phenotype, however, are LW–.
In 1981, a new blood group antigen, Nea was reported
with a frequency of approximately 5% in the Finnish population. Anti–Nea
displayed a variation in strength of reactivity similar to anti–LW that
suggested a relationship between Nea and LW. It is now known that Nea
and LW are products of allelic genes. Therefore, the antigen formerly called LW
had become LWa and Nea becomes LWb.
****** ANTIBODIES OF THE RH SYSTEM ******
1.
The majority of the Rh antibodies are IgG
(7S) reacting at 37oC, though in the early stages of the immune
response, saline agglutinins maybe prominent (i.e., IgM).
2.
Saline agglutinins are known to be IgM and do
not cross the placenta. IgG Rh antibodies do not agglutinate saline suspended
Rh–positive cells, though they will agglutinate cells suspended in concentrated
bovine albumin.
The indirect antiglobulin test is
a very sensitive way of demonstrating reactivity between IgG anti–D and Rh
positive red cells. A technique with enzyme treated cells is, in many cases,
even more sensitive.
3.
IgG antibodies of almost any specificity are
capable of crossing the placental barrier and commonly cause hemolytic disease
of the newborn. Anti–D is the antibody most often implicated as cause of this
condition.
4.
Rh antibodies are capable of causing severe
hemolytic transfusion reactions.
5.
Naturally occurring anti–D is extremely rare,
though many examples of naturally occurring anti–E are known. Anti–Cw
has also been known to occur naturally.
6.
Rh antibodies are most commonly stimulated by
transfusion or pregnancy; they generally develop eight to nine weeks after
transfusion, and sometimes much later.
7.
Rh antibodies do not bind complement, because
the triggering of the complement cascade calls for at least two IgG molecules
to be bound to the cell in close proximity (i.e., juxtaposition). The condition
is not fulfilled with Rh antibodies because the antigen receptors are too far
apart.
8.
The antibodies most commonly found in
immunized patients are anti–D and anti–G.
Rh Immune Globulin (RhoGAM)
The Rh immune globulin is a purified gamma globulin (IgG) containing
anti–Rho (D) antibody which when administered (within 72 hours) to
unsensitized Rh negative mothers who deliver Rh–positive babies suppressed Rho
(D) alloimmunization. It also suppresses antibody production in cases where
Rh–negative individuals have been transfused with Rh–positive blood.
Categories of Rh antibodies
1.
Complete antibodies or bivalent antibodies or
saline agglutinins
These are antibodies which could
produce agglutination in saline medium but weakened or destroyed by heating
over 60oC. They rarely cross the placenta (to cause HDN). They are usually IgM
in nature.
2.
Incomplete antibodies or univalent antibodies
or serum albumin agglutinins
These are antibodies that react
with, but fail to cause visible agglutination on a saline suspension of red
cells possessing the corresponding antigenic determinant. They are usually IgG
in nature thus causing HDN. They produce agglutination only under the following
conditions:
a.
Suspension of RBC in albumin
b.
Addition of Coomb’s reagent
c.
Enzyme treatment of RBC
Causes of absence of
agglutination:
a.
Location of antigenic determinants on the red
cell surface.
b.
Size and configuration of antibody molecule.
c.
Physico–chemical properties of the
suspending medium.
Synonyms:
a.
Cryptoagglutinoids
b.
Thermostable agglutinins
************ Rh TYPING ************
Since anti–D is not normally present in the serum of D–negative
people, “reverse grouping” cannot be done for Rh as with ABO. For this reason,
it is recommended that Rh tests be done in duplicate using two different
technique or be done independently by two technologist.
Rh positive donor blood, erroneously typed as negative, will not
be detected by compatibility testing unless the potential recipient has already
produced anti–D. Similarly, the crossmatch will be compatible if an Rh negative
patient has been falsely typed as Rh positive and Rh+ donor has been selected. The entire burden of preventing the formation of
anti–D through transmission lies in accurate testing of the red cells of patients
and donor, a compatible crossmatch does not preclude immunization to D or other
antigens.
If anti–D is already present in the serum of an Rh negative
patient, it is even more essential that Rh typing of potential donors be
correct. Administration of D positive blood to a patient whose serum contains
anti–D may be fatal.
Reagents for Rh typing contain antibodies with the same
specificity but different reactivity. Anti–D saline agglutinins are used to prepare
the reagent labeled anti–Rho (anti–D) serum for Saline Tube Test;
therefore the antiserum agglutinates saline suspended D positive cells at 37oC.
Anti–D albumin agglutinins are used for the reagent labeled anti–Rho
(anti–D) serum for Slide and Modified Tube Test and albumin is incorporated
into the reagent.
Techniques used in Rh typing
Reagent Method Cell suspension Incubation
Anti–Rho (anti–D) Saline Saline 37oC – 1 hour
Serum for saline Tube
test suspended cells
Test tube
Anti–Rho (anti–D) Slide
test whole 45oC–2 mins.
viewbox
Serum for Slide and
Modified Tube Test
Modified
Tube serum suspended immediate
Test cells centrifugation
saline
suspended immediate
cells centrifugation
The Saline Tube Test
This is preferred by many workers because washed red cells are
used and, thus, interfering factors that may be present in the patient’s serum
are eliminated. A bovine albumin control is essential in Rh typing with serum
suspended cells, is unnecessary when the saline tube test is used. The saline
tube test is particularly valuable for typing cells of patients with a positive
direct antiglobulin test due to autoantibodies. Such cells are often seen in
patients with acquired hemolytic anemia; the antibodies may be specific or
nonspecific. The globulin “coating” the cells does not interfere with the
detection of the D antigen by the anti–Rh (anti–D) serum for saline tube test and tests for other
antigens are usually reliable if saline tube methods are used.
Cells sensitized with anti–D in vivo, such as cells of babies with hemolytic disease due to anti–D,
may not type correctly by any direct antiglobulin method because all or almost
D antigen sites maybe blocked. When all the D antigen sites on the cells take
anti–D in vivo, no sites are available for the attachment of the reagent anti–D
and the cells appear to be Rh negative. If an eluate prepared from sensitized
red cells shown to contain anti–D, the red cells are then established as Rh
positive; were they really Rh negative, they would not absorb anti–D in vivo
and eluates could not contain the antibody. Cells of babies with HDN due to
saline tube test method, but other methods or Rh typing are no more reliable in
these cases.
Procedure for Saline Tube Test
1.
Prepare a 2% suspension in saline of washed
cells from freshly drawn whole blood.
2.
Add 1 drop of anti–Rh saline tube test serum
to a small test tube.
3.
Add 2 drops of the 2% cell suspension.
4.
Incubate at 37oC for 60 minutes.
5.
Examine sedimented cells for agglutination.
6.
If there is no agglutination, shake the tube
and centrifuge at 1000 – 2000 rpm for 1 minute.
7.
Resuspend the cells and examine for
agglutination. If desired, reading maybe confirmed with the aid of a small hand
lens or magnifying mirror.
The Slide and Modified tube test
The same anti–D reagent is used for both the slide test and the
modified tube test. The slide test uses whole blood, i.e., red cells suspended
in serum or plasma. The modified tube test may be done with cells suspended in
serum, plasma or saline. The slide test and modified tube test using serum
suspended cells are essentially the same test, with the same advantages and
disadvantages. The sue of saline suspended cells for the modified tube test
provides many but all the advantages of the saline tube test. In addition, the
modified tube test can be performed more rapidly.
Serum suspended cells used in either the slide or modified tube
test may give false reactions if the serum is abnormal in some respect. Cells suspended
in abnormal serum and subjected to heat may aggregate spontaneously and appear
to be agglutinated (pseudoagglutination). Bovine albumin in the test reagent
tends to enhance the possibility of false reactions in Rh typing of patient
with abnormal serum proteins.
Slide testing
False positives:
1.
If albumin control is positive, disregard
results and repeat test with saline– active reagents.
2.
Small fibrin clots may give the appearance of
agglutination.
3.
Blood incompletely anticoagulated may clot on
the heated slide.
False negatives:
1.
Too weak cell suspension may agglutinate
poorly. Whole blood from severely anemic patients may not be sufficiently
concentrate cell suspension.
2.
Cells in a saline suspension react poorly or
not at all.
3.
Weakly active cells may take the full 2
minutes to agglutinate. Do not expect reactions to be as rapid as with ABO
reagent.
4.
Failure to identify reagents at time of sue
may allow albumin antiglobulin serum or some other colorless reagent to be
added instead of anti–Rho (D).
Tube testing
False positives:
1.
If cells and serum remain together too long
before test is read, the high protein medium may produce rouleaux which
resembles agglutinates.
2.
If a combined antibody is used instead of
anti–Rho (D), cell which lack Rho (D) but contain the other antigen may be
agglutinated.
3.
Some specificity other than anti–Rho (D) may
be present in the serum.
False negatives:
1.
If cells and serum remain together too long
before tests are read, antibody may elute from weakly reactive cells and small
agglutinates may disperse.
2.
Failure to identify reagents at time of use may
result in albumin, antiglobulin serum or some other colorless reagent being
added instead of anti–Rho (D).
3.
Inadvertent failure to add reagent will give
smooth suspension of cells.
Procedure of Slide Test
1.
Prepare a 40–50% suspension of cells in their
own or group compatible serum or whole blood may be used.
2.
Add 2 drops of the cell suspension to a slide
pre–warmed to 40–50oC on a slide viewing box.
3.
Add 1 drop of anti–Rh slide test serum.
4.
Mix well and tilt the view box back and
forth.
5.
Examine for agglutination. Interpret as
negative those cells which are not agglutinated in 2 minutes. Do not observe
longer than 2 minutes.
Procedure for modified tube test
1.
Prepare a 2% suspension of cells in their own
group compatible serum.
2.
Add 1 drop of anti–Rh slide test serum to a
small test tube.
3.
Add 2 drops of 2% cell suspension.
4.
Mix and centrifuge at 1000 – 2000 rpm for 1
minute.
5.
Examine for agglutination.
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