In 1939, Hemolytic Disease of the Newborn was first
described by Levine and Stetson. The cause of hemolytic disease of the
cause was not specifically identified but maternal antibody suspected. A
year later (1940) Karl Landsteiner and Alexander Wiener injected animals with Rhesus monkey
cells to produce an antibody which reacted with 85% of human red cells,
which they named anti-Rh. Within a year Levine made connection between maternal antibody causing HDN
and anti-Rh. Between 1943-45 the other common antigens of the Rh system
were identified. For many years the exact inheritance of the Rh
factors were debated Weiner promoting Rh and hr terminology and
Fisher-Race utilizing DCcEe for the various Rh antigens. In 1993,
Tippett discovered true mode of Rh inheritance using
D (Rho) is the most important antigen
after A and B antigens. Unlike the anti-A and anti-B antibodies,
anti-D antibodies are only seen if a patient lacking D antigen is
exposed to D + cells. The exposure of D+ cells usually occurs
through pregnancy or transfusion.
There are 5 principle antigens that may be found in
most individuals. They are:
- D found in 85% of the population
- C found in 70% of the population
- E found in 30% of the population
- c found in 80% of the population
- e found in 98% of the population
- (d) which has never been identified but refers to
the 15% of the population who has no D antigen
There are at over 50 Rh antigens that have been
identified including those that are either combinations of these antigens
or weak expressions of the above antigens, but most Rh problems are due to
D, C, E, c or e.
The common alleles are:
- C and c are alleles with Cw occasionally
seen as a weaker expression of C.
- E and e are alleles although E is seen only a
third as often as e. The e antigen is referred to as a high incidence
antigen since it is found in 98% of the population.
- D and the lack of D (or d) are alleles.
Characteristics of Rh antigens
The Rh antigens together are proteins of 417 amino
acids. These proteins cross the red cell membrane 12 times. There
are only small loops of the protein on the exterior of the cell
Therefore the Rh antigens are not as available to react with their
specific antibodies and there are fewer antigen sites than ABO.
Unlike the ABO system the Rh antigens are not soluble and are not
expressed on the tissues. They are well developed at birth and
therefore can easily cause hemolytic disease of the newborn if the baby
has a Rh antigen that the mother lacks. Besides the antigens being
well-developed at birth, they are very good immunogens. This is
especially true to D, which if the most immunogenic after A and B
Unlike the ABO antibodies that are mainly IgM, the
Rh antibodies are commonly IgG. They are NOT naturally occurring and
therefore are formed by immune stimulus due to transfusions or baby's red
blood cells during pregnancy. The most common antibody to form is
anti-D in Rh negative individuals.
Since Rh antibodies are IgG they bind best at 37oC
and their reactions will be observed with the indirect antiglobulin
technique. Agglutination reactions are enhanced by high protein (albumin),
low-ionic strength saline (LISS), proteolytic enzymes (ficin) and
polytheylene glycol (PEG).
Rh antibodies will react more strongly with
homozygous cells than with heterozygous cells. For example, an
anti-E will react with strongly with E+E+ cells and more weakly with E+e+
cells. This is called dosage.
Both Hemolytic Disease of the Newborn and Hemolytic Transfusion Reactions
can occur due the various Rh antibodies. Anti-D has been the biggest
concern since it was recognized in the 1940's as being the most common
cause of hemolytic disease of the newborn. Since the D antigen is so
immunogenic we screen all donor units for the D antigen. Therefore
if an individual is A+, it means both the A and the D antigens are
present. On the other hand, if an individual is A-, the A antigen is
present and the D antigen is absent.
To prevent problems due to anti-D:
- we try to always give Rh-negative individuals
- and we give Rhoimmune globulin to
Rh-negative mothers to prevent the formation of anti-D during pregnancy.
The incidence of Rh antibodies
- Anti-D most common antibody seen in Rh(D) negative people
- Anti-E most common antibody seen in Rh pos people
since only 30% of the population have the antigen
- Anti-C or Anti-c less common - most people have the antigen
- Anti-e often seen as autoantibody and will make
it difficult to find compatible blood since 98% of the population have
the e antigen
- Anti-C,e or Anti-c,E often seen in combination.
If a patient lacks both a C and e and has made an anti-C, then
enhancement techniques should be done to make sure that an anti-e is not
Rh System Inheritance
From the 1940's to the 1990's the mechanism for
inheritance of the Rh Blood Group System was in question. The
terminology that is part of the Fisher-Race Theory is most commonly used
The Fisher-Race theory involved the presence of 3
separate genes D, C, and E and their alleles c and e and the absence of D
since an anti-d has never been found. These three genes are closely
linked on the same chromosome and are inherited as a group of 3. The
most common group of 3 genes inherited is CDe and ce (D negative) is the
second most common.
Weiner believed there was one gene complex with a
number of alleles resulting in the presence of various Rh antigens.
According to Weiner there were 8 alleles, Ro, R1, R2,
Rz, r, r', r", ry , which
ended up with different antigens on the red cells that he called Rho,
rh', rh", hr', hr". Weiner terminology is not use as often today,
but you will often see Rho(D) when a person considered to be
Rh-positive. At times the gene terms are easier to use than
Fisher-Race. If a person has the Fisher-Race genotype of DCe/DCe, it
is easier to refer to that type as R1R1
2. Made up of combinations of genetic products
In 1986, Tippett predicted that there are two closely-linked genes -
RHD and RHCE. The RHD gene determines whether the D antigen that
spans the membrane is present. Caucasians who are D negative have no gene
at that gene loci. In the Japanese, Chinese, and Blacks of African
descent have an inactive or partial gene at this site.
The RHCE gene determines C, c, E, e antigens
produced from the alleles:
Rh Gene Complexes, Antigens,
Possible Combinations and Percentages
(more common in Blacks)
Translating From Wiener To Fisher-Race
There are times when you will need to convert Weiner
to Fisher-Race or vice versa. It will be easier to do these
conversions if you remember the following:
- R always refers to D whether it is Ro,
R1, R2, or the very rare Rz.
- r always refers to the lack of D
- o refers to having no C or E
- 1 or ' always refers to C
- 2 or " always refers to E
- The very rare haplotypes that have both a C and E
are given letters from the end of the alphabet z and y.
Determining Genotypes From Phenotypes
The following steps will be helpful in determining
from the individual's phenotype. These rules are based on
probability so the least likely genotypes will involve Rz or ry.
- Type patient for the five Rh antigens: D, C, c, E, e
- Start with D: is it positive or negative?
- If negative, the individual will be homozygous
- If positive for D, you can't tell yet whether
the individual is homozygous or heterozygous for D. Therefore,
put D on just one chromosome.
- Look at C: is it positive or negative?
- If negative, put c on each chromosome.
- If positive, look at c result to determine if
the C is homozygous or heterozygous. If there is no c present,
there would be two C and it would be homozygous. If a c is
present as well as C, they are heterozygous.
- If homozygous, then put C on each chromosome.
- If heterozygous, put C on same chromosome as D;
put c on other.
- Look at E: is it positive or negative?
- If negative, put e on each chromosome.
- If positive, look at e result to determine if
homozygous or heterozygous.
- If homozygous, put E on each chromosome.
- If heterozygous, put E on same chromosome as
the D unless the D already has a C; put e on other chromosome.
DCe is more common than DcE and DCE is extremely rare.
Put C and E together on same chromosome only if no other possible
Most Common Genotypes
The following genotypes are listed as the most
common with 1 being the most common in Whites and 7 the least common.
Rz and ry are so rare they are not included in the
Incidence of the most common
D, C, c, e
D, C, e
Applications of Rh genotyping
of the blood group antigens is based on a process of exclusion.
Since the RHD and RHCE are closely linked and Ce, ce, cE are
produced by a single gene, there are limited combinations that the
father can provide.
by testing the father's Rh genotype. This helps predict likelihood of HDN due to D when mom has anti-D.
The most common Rh genotype of the father will indicate whether the baby
has O%, 50%, or 100% probability of being D positive.
- If the father is also D negative (ce/ce), the
baby will be D negative as well and there is a 0% probability of the
baby suffering from Rho HDN.
- If the father's Rh genotype appears to be either,
R1r, R2r or Ror, the baby has a 50% probability
of being D positive and suffering from Rho HDN.
- On the other hand if a father's Rh genotype
appears to be any of the following, R1R1, R2R2,
R1R2, RoRo, R1Ro,
or R2Ro, the baby has a 100% probably of getting
a D gene from his father and therefore being D positive and
suffering from Rho HDN.
Weak D (Du)
Weak D is a weakly expressed D antigen that will
only be demonstrated after incubation at 35-37oC followed
with antiglobulin testing. (ie being demonstrated only by Coombs technique).
An Rh control must always be run along with the weak D test.
Always consult the product insert to determine if Rh Control needs to be
run when performing the immediate spin D testing. The
following results could be obtained when performing the D testing:
any time the Rh control is positive, you can not interpret the
results and need to perform further testing
Testing for Weak D
AABB requires that all donor blood that originally
fails to react with anti-D at immediate spin must be tested for weak
D. Units that test weak D positive would be labeled D positive and
would be transfused only to D positive individuals.
Hospitals may or may not test all Rh negative recipients for weak D.
The cost of time and reagents is minimized if only the immediate
spin. This may create some confusion with the recipient if their
donor card indicates they are Rh positive but they type Rh negative when
they are the recipient. Recipients that type D negative at immediate
spin would be given D negative blood, which not create a problem for the
When performing testing prenatal and postnatal
mothers, D-negative blood at immediate spin would be tested for weak D
as well to determine if they are eligible for Rho Immune
Globulin. Since Rho Immune Globulin is actually anti-D it
is safe for a true D negative, but not for a weak D positive mother.
Why do weak D's exist?
There are three explanations for weak D's.
- Quantitative Weak D
There are individuals that quantitatively produce fewer D antigen sites.
This is more common in Blacks and is often seen with the Dce haplotype.
On rare occasions among Whites an unusual DCe or DcE may also produce a
quantitatively decrease weak D.
- Position Effect Weak D In this case the D
is weakened by the position of a C on the opposite haplotype which is
called the trans position. The two Rh genotype combinations where
this type of weak D is seen are: Dce/Ce and DcE/Ce. Today
this type of weak D would type as a regular D due to the improvement of
- Partial D antigen
It has been found that some D-positive individuals make an alloanti-D
that reacts with other D positive cells but not their own. Many of
these will demonstrate a weak D type of reaction. In this type of weak
D, the individuals are lack some of the components of the D antigen and
therefore are able to make allantibodies to those specific components if
they are transfused with D positive blood.
Other Rh System Variants
There are presently 46 Rh antigens identified and
named. The following are the most common of those variants
is a low frequency antigen found in approximately 2% of Whites and
1% of Blacks. It is not an allele of C and c. Its allele is
MAR, which is found in 99.9% of the population.
- V and VS are
low frequency alleles found in 1% or less of the Whites, but are more
common in Blacks. V is found in 30% of the Blacks and VS in 32%.
- G is present
when D or C present due to the present of serine at the 103 position of
the Rh polypeptide. Anti-G will react with both D+ and C+ cells.
- f is present
when c and e together on same chromosome: Dce or ce. This is the
most common of what are called cis product antigens.
has no Rh antigens on their red cells but these individual can
transmit normal Rh antigens to their offspring. In the most common type
the core Rh polypeptide is missing. A less common type has the regulator
gene that turns off the expression of Rh. There have been at least
43 individuals in 14 families that are Rhnull. In
these individuals the red blood cell membrane is abnormal and some of
these have been identified when it was observed that they had hemolytic
anemia and abnormal red cell morphology. If these individuals
develop an Rh antibody following a transfusion or pregnancy, it is
considered a anti-total Rh antibody.
False positive D's occur:
- When following through to AGT for weak D and will
be identified as false positive by a positive Rh control. This is
seen when a patient/donor has strong positive DAT. The cells are coated with antibody (not necessarily Rh antibody) in vivo. Albumin
is necessary in reagent Anti-D to overcome the zeta potential
allowing cells coated with IgG Anti-D to get close enough together to
agglutinate, but cells coated in vivo with any IgG antibody will also agglutinate
These false positives are corrected by using form of Anti-D that does
not require albumin. There are two types of alternative types of
- Monoclonal (IgM) anti-D will cause agglutination of
D positive cells without the presence of albumin at room temperature.
A number of facilities normally use this type of anti-D and therefore do
not routinely use Rh control.
- Chemically modified anti-D has been modified by
breaking the disulfide bonds closest to the hinge region so antibody can
reach cells that are farther apart.
- False positive can also be caused by rouleaux
formation, which will look like agglutination macroscopically.
Rouleaux would be identified microscopically due to the "coin-stacking"
appearance of the red cells. This false positive would be corrected
by washing cells 3 to 4 times and then retesting.
False negatives are not readily identifiable, but
can occur in the following instances:
- The most common is the result of too heavy cell
suspension due to too many cells for the amount of antibody in the
- They may also rarely be caused by extremely
strong positive DAT. In this case a the patient's D antigen sites are
coated in vivo and there are no sites left for commercial anti-D to
attach to. This can be fixed by heating cells gently to elute off
antibody without damaging cells, then re-test.
OBJECTIVES - Rh SYSTEM
- Briefly describe how and when the Rh system was discovered
- List the major Rh antigens and state the frequency each is seen in
the Caucasian population.
- Describe the characteristics common to the major Rh antigens and
compare them to the ABO system.
- Explain the Tippett theory of inheritance.
- For any given Rh phenotype, predict the most likely genotype in both
the Wiener and Fisher-Race nomenclatures.
- For any given Wiener genotype, list the Rh antigens present.
- Explain why Rh genotyping is important.
- Give three explanations for the weak D phenotype.
- Discuss how the weak D phenotype applies to donors, recipients, and
- State the relative frequencies of the Cw, V and VS antigens.
- Explain the G, f, and Rh null phenotypes.
- Describe characteristics common to antibodies in the Rh system.
- List the more common antibodies seen in the Rh system.
- Discuss the use of albumin and enzymes in identifying Rh antibodies.
- Explain how false positives can occur when testing for the Rh
antigens, and describe how the problem may be overcome.
- Explain how false negatives can occur when testing for the Rh
antigens, and describe how the problem may be overcome.
- Differentiate between high-protein anti-D, chemically modified
anti-D, and saline anti-D.
- Correctly perform, interpret, and record the Rh type of any given
- Recognize when chemically modified or saline Rh reagents must be
- Correctly perform, interpret, and record a weak D (Du)test.
- Correctly perform, interpret, and record the Rh phenotype of any
given sample, and state its most likely genotype.