Rh Blood Group System - Definition, Antigens, Antibody

Rh blood group is a human blood group system. It is one of the most important blood group after ABO blood group. It has great importance in blood transfusion and pregnancy.

The Rh blood group is mainly identified by Rh factor. This Rh factor is the D antigen. It is present on the surface of red blood cells.

If D antigen is present on RBC, the blood is called Rh positive. If D antigen is absent, the blood is called Rh negative. For this reason blood group is written as A+, B+, AB+, O+ or A-, B-, AB-, O-.

The Rh system contains many antigens. More than 50 Rh antigens are present. But the important antigens are D, C, c, E and e. Among these, D antigen is the most important antigen.

The Rh antigens are pure transmembrane proteins. They are present inside the membrane of red blood cells. They help in keeping the shape and flexibility of the cell. They may also help in transport of ammonium and carbon dioxide.

The Rh antigens are controlled by genes. These genes are present on chromosome 1. The RHD gene forms RhD protein and carries D antigen. The RHCE gene forms RhCE protein and carries C, c, E and e antigen.

Rh blood group is highly immunogenic. It means its antigen can easily produce immune reaction. When Rh negative person receive Rh positive blood, antibody may be formed against Rh antigen.

This reaction may destroy the red blood cells. This is called hemolytic transfusion reaction. It may be severe and dangerous.

In pregnancy, Rh factor is also very important. If mother is Rh negative and fetus is Rh positive, then mother can form Rh antibody. This antibody may pass through placenta.

After crossing placenta, it destroys fetal red blood cells. This condition is called hemolytic disease of fetus and newborn (HDFN).

For this reason, Rh typing is done before blood transfusion. It is also done in pregnant mother. It helps to prevent transfusion reaction and fetal disease.

Antigens of The Rh Blood Group

Rh blood group system contains 56 recognized antigens. These antigens are present on the surface of red blood cells. Among them some are common and some are rare.

The most important Rh antigens are D, C, c, E and e. These antigens are mainly important in blood transfusion and pregnancy.

Major and clinically important antigens

D antigen (RH1)
D antigen is the most important antigen of Rh blood group. It decides the blood group as Rh positive or Rh negative. There is no d antigen. Small d only means absence of D antigen.
C antigen (RH2)
C antigen is another important antigen of Rh system. It is clinically significant and may produce antibody after incompatible transfusion.
E antigen (RH3)
E antigen is also an important Rh antigen. It can take part in immune reaction and transfusion problem.
c antigen (RH4)
c antigen is clinically important antigen. It is present in many individuals and may cause antibody formation.
e antigen (RH5)
e antigen is one of the five main Rh antigens. It is also considered during blood grouping and antibody detection.

Other commonly typed antigens

Cw antigen (RH8)
Cw antigen is a common typed antigen of Rh blood group. It is not as important as D antigen, but it may be tested in special cases.
Cx antigen (RH9)
Cx antigen is another antigen of Rh system. It is less common than the main antigens.
V antigen (RH10)
V antigen is a Rh antigen. It may be present in some red blood cells and has transfusion importance.
VS antigen (RH20)
VS antigen is also included in commonly typed Rh antigens. It is checked in selected cases.

Compound antigens

Compound antigens are formed when some Rh alleles are present on same chromosome. This condition is called cis position.

f antigen (ce / RH6)
f antigen is a compound antigen. It is formed by c and e present together.
Ce antigen (RH7)
Ce antigen is formed when C and e are inherited together.
CE antigen (RH22)
CE antigen is formed from C and E combination.
cE antigen (RH27)
cE antigen is formed from c and E combination.

Shared antigens

Shared antigens are encoded by either RHD or RHCE gene loci. They may be present with more than one Rh antigenic form.

G antigen (RH12)
G antigen is present on almost all red cells which have D antigen or C antigen. It is important because it may look like anti-D and anti-C reaction.
FPTT antigen (RH50)
FPTT antigen is a shared antigen of Rh system.
BARC antigen (RH52)
BARC antigen is included under shared Rh antigens.
DAK antigen (RH54)
DAK antigen is another shared antigen of Rh blood group.
Rh29 antigen
Rh29 is a high frequency antigen of the Rh system.
CEWA antigen (RH62)
CEWA antigen is also included in this group.

Less common and rare antigens

The following are less common and rare Rh antigens.

Ew antigen (RH11)
Hr0 antigen (RH17)
Hr antigen (RH18)
hrS antigen (RH19)
CG antigen (RH21)
Dw antigen (RH23)
c-like antigen (RH26)
hrH antigen (RH28)
hrB antigen (RH31)
Rh32 antigen
Rh33 antigen
HrB antigen (RH34)
Rh35 antigen
Bea antigen (RH36)
Rh39 antigen
Tar antigen (RH40)
Rh41 antigen
Rh42 antigen
Crawford antigen (RH43)
Nou antigen (RH44)
Riv antigen (RH45)
Sec antigen (RH46)
Dav antigen (RH47)
JAL antigen (RH48)
STEM antigen (RH49)
MAR antigen (RH51)
JAHK antigen (RH53)
LOCR antigen (RH55)
CENR antigen (RH56)
CEST antigen (RH57)
CELO antigen (RH58)
CEAG antigen (RH59)
PARG antigen (RH60)
CEVF antigen (RH61)
CETW antigen (RH63)

These rare antigens are not typed routinely. They are mainly important in special transfusion cases, antibody investigation and rare blood group studies.

Importance of The Rh Blood Group System

The following are the importance of Rh blood group system–

Transfusion– Rh blood group is very important in blood transfusion. It is the second important blood group after ABO blood group. Mainly D antigen is highly immunogenic.
Mismatch reaction– If Rh negative person receive Rh positive blood, then antibody may be formed. These antibodies destroy transfused red blood cells. This is called hemolytic transfusion reaction.
Pregnancy– Rh blood group is important in pregnancy. If mother is Rh negative and fetus is Rh positive, then mother may form anti-D antibody. These antibody can cross the placenta.
HDFN– The anti-D antibody destroy fetal red blood cells. This causes hemolytic disease of fetus and newborn (HDFN). It is also called erythroblastosis fetalis.
Fetal damage– In severe condition, HDFN may cause severe anemia in fetus. It may also cause brain damage and death of fetus or newborn.
RhIG– Rh immune globulin (RhIG) is given to Rh negative mother. It prevents formation of anti-D antibody. RhoGAM is one example of RhIG.
Screening– Rh typing is done routinely in blood donors, transfusion patients and pregnant women. It helps to know Rh(D) status. It prevents transfusion and pregnancy complication.
RBC structure– Rh proteins are present in red blood cell membrane. They help to maintain biconcave shape and flexibility of RBC. They also maintain structural integrity of cell.
Rh-null– In rare condition, Rh proteins are completely absent. This is called Rh-null phenotype. In this condition RBCs become abnormal in shape and chronic hemolytic anemia may occur.
Transport– Rh protein complex may act as transport channel. It helps in movement of ammonia across RBC membrane. It has role in nitrogen metabolism and pH regulation.
Infection– Some studies suggest that Rh membrane complex may affect entry of some viruses. It may interact with viral surface protein. So Rh phenotype may influence susceptibility to infections like SARS-CoV-2.

Genetic Basis of The Rh Blood Group System

The following are the genetic basis of Rh blood group system–

Rh blood group system is mainly controlled by two closely linked genes. These are RHD gene and RHCE gene. Both genes are present on chromosome 1.
RHD and RHCE genes are highly similar genes. They are present very close to each other. They control the formation of main Rh antigens.
RHD gene is originated from duplication of ancestral RHCE gene during mammalian evolution. For this reason both genes show much similarity in structure.
Both RHD and RHCE genes are about 97% identical. Each gene contain 10 exons. They cover about 75-kb DNA region.
These two genes are arranged in back-to-back manner. They are present in opposite direction. A small intervening gene called SMP1 is present between them.
RHD gene produces RhD protein. This protein carries D antigen. D antigen is the most immunogenic antigen of Rh blood group system.
Rh negative phenotype means absence of D antigen on the surface of red blood cells. It is mainly due to changes or absence of RHD gene.
In European population, Rh negative phenotype is mostly caused by complete deletion of RHD gene. This deletion occurs due to genetic recombination.
In African and Asian population, Rh negative phenotype may occur due to RHD pseudogene. It may also occur due to RHD hybrid gene.
In RHD pseudogene, premature stop codon is present. This stop codon stops the formation of normal RhD protein.
RHD hybrid gene contains mixed sequence of RHD and RHCE genes. So normal D antigen may not be formed.
RHCE gene codes for C, c, E and e antigens. These antigens are also present on red blood cell membrane.
The difference between C antigen and c antigen is due to four single nucleotide polymorphisms (SNPs). The main change is amino acid change at position 103.
At position 103, Serine is present for C antigen. Proline is present for c antigen.
The difference between E antigen and e antigen is due to one SNP. This causes amino acid substitution at position 226.
At position 226, Alanine is present for e antigen. Proline is present for E antigen.
Another gene called RHAG is also needed for Rh system. It is present on chromosome 6. It is essential for proper expression of Rh proteins.
RHAG gene encodes Rh-associated glycoprotein (RhAG). This protein works as molecular chaperone. It helps to transport Rh proteins to red blood cell membrane.
If mutation silence RHAG gene, then Rh proteins cannot reach the cell surface. In this condition all Rh antigens are absent.
This condition is called regulator form of Rh-null phenotype. It is a rare condition where Rh antigens are missing from red blood cells.

Rh Antigens and Their Characteristics

The following are the Rh antigens and their characteristics-

Rh blood group system has 56 recognized antigens. These antigens are present on red blood cells. They are important in blood transfusion and pregnancy.
Rh antigens are protein antigens. They are not sugar type antigen like ABO blood group. They are non-glycosylated transmembrane proteins.
These antigens are present in RBC membrane. They pass through the membrane about 12 times. They are expressed mainly on cells of erythroid line.
Rh proteins help to maintain the structure of red blood cell membrane. They help in cell shape and flexibility. They may also help in transport of ammonium or carbon dioxide.
The main Rh antigens are D, C, c, E and e. These are the most clinically important antigens. They can form antibodies after incompatible transfusion.
D antigen (RH1) is the most important Rh antigen. It is highly immunogenic. Presence of D antigen makes blood Rh positive.
Absence of D antigen makes blood Rh negative. There is no real d antigen. Small d only means absence of D antigen.
D antigen is encoded by RHD gene. This gene forms RhD protein. This protein is present in RBC membrane.
C antigen (RH2), c antigen (RH4), E antigen (RH3) and e antigen (RH5) are other major Rh antigens. These are encoded by RHCE gene.
The frequency of C, c, E and e antigens are not same in all population. e antigen is very common. It is present in about 98% of Caucasian and African descent individuals.
Some Rh antigens are compound antigens. They are formed when two specific alleles are present on same chromosome. This is called cis position.
f antigen (RH6) is a compound antigen. It is formed when c and e alleles are inherited together on same haplotype.
Other compound antigens are Ce (RH7), CE (RH22) and cE (RH27). These are also formed by special combination of Rh alleles.
Some Rh antigens are shared antigens. These can be encoded by either RHD or RHCE gene. They are not limited to only one gene.
G antigen (RH12) is a shared antigen. It is present on almost all red cells having D antigen or C antigen. It is absent when both D and C are absent.
Some Rh antigens are low incidence antigens. They are found only in small number of people. They may still cause transfusion problem.
Cw antigen (RH8) is a low incidence antigen. It is found in about 2% of Caucasians. It is very rare in people of African descent.
V antigen (RH10) and VS antigen (RH20) are present in about 30% of African Americans. These antigens are rare in Caucasians.
Weak D is a variant form of D antigen. In this condition, D antigen is complete but present in low amount on cell surface. It is due to amino acid change in transmembrane region.
Partial D is another variant form of D antigen. In this condition, one or more outer epitopes of D antigen are absent. These persons can form antibody against the missing part of D antigen.
Del phenotype is very weak expression of D antigen. It is not detected by routine method. It is detected by special laboratory techniques like adsorption and elution.
Rh-null phenotype is very rare condition. It is also called Golden Blood. In this condition all Rh antigens are absent from red blood cells.
In Rh-null phenotype, RBCs become abnormal in shape. This may cause stomatocytosis and mild to moderate hemolytic anemia.

Structure and Expression of Rh Antigens on Red Blood Cells

The following are the structure and expression of Rh antigens on red blood cells–

Rh antigens are protein type antigens. They are not carbohydrate or sugar type antigens like ABO blood group. They are non-glycosylated and hydrophobic proteins.
Each Rh protein contains 417 amino acids. These amino acids form the membrane protein structure. This protein is present in red blood cell membrane.
Rh proteins are integral transmembrane proteins. They pass through the phospholipid bilayer of RBC membrane for 12 times.
This arrangement forms 6 extracellular loops. These loops are present outside the cell. The antigenic parts are mainly exposed in these extracellular loops.
It also forms 5 intracellular loops. These loops are present inside the cell. Both N-terminal and C-terminal ends remain inside the red blood cell.
Rh proteins do not work alone in RBC membrane. They are associated with Rh-associated glycoprotein (RhAG). RhAG is very important for expression of Rh antigens.
Rh proteins and RhAG together form a membrane complex. This is called Rh core complex. It is present in the membrane of red blood cells.
The Rh core complex is believed to be a tetramer. It contains two RhAG molecules and two Rh proteins. These Rh proteins may be RhD, RhCE or both.
The Rh core complex is attached with the underlying cytoskeleton of RBC. This attachment helps to maintain shape and flexibility of red blood cells.
This complex interacts with other membrane proteins. These include Band 3, CD47, glycophorin B and LW glycoprotein.
Due to this membrane attachment, Rh proteins help in structural integrity of RBC membrane. They help the cell to keep biconcave shape and normal deformability.
Rh antigens are expressed only on cells of erythroid line. It means they are mainly found on red blood cells. They are not generally expressed on other body cells.
RhAG is encoded by RHAG gene. This gene is present on chromosome 6. It works as required factor for stable expression of Rh antigens.
Without RhAG, RhD and RhCE proteins cannot reach the red blood cell membrane. So Rh antigens are not properly expressed on the cell surface.
Mutation in RHAG gene may stop the expression of Rh proteins. In this condition all Rh antigens may be absent from RBC membrane.
This condition is called regulator type Rh-null phenotype. It is a very rare condition. In this condition red blood cells completely lack Rh antigens.

Rh Positive and Rh Negative Blood Types

Rh positive blood type

Rh positive blood type means presence of Rh(D) antigen on the surface of red blood cells. This antigen is also called D antigen.
If a person has A+ blood group, then the RBC has A antigen and Rh(D) antigen. So the blood type is called Rh positive.
Rh positive blood type is most common in human population. It is found in about 94% of world population.
The frequency of Rh positive blood type is different in different population. It is about 85% in Caucasians, 92% in African descent people and 99% in Asians.
Rh(D) antigen is inherited as dominant genetic character. So if D antigen is expressed on RBC, the person becomes Rh positive.

Rh negative blood type

Rh negative blood type means complete absence of Rh(D) antigen on the surface of red blood cells. The RBC does not carry D antigen.
If a person has A- blood group, then the RBC has A antigen but does not have Rh(D) antigen. So the blood type is called Rh negative.
Rh negative blood type is less common than Rh positive blood type. It is found in about 6% of world population.
The frequency of Rh negative is more in Caucasian population. It is about 15% in Caucasians.
Rh negative is less common in African descent people. It is found in about 8% of them. It is very rare in Asians, about 1%.
Being Rh negative is only a genetic variation. It does not affect general health or well-being of a person.
In Caucasians, Rh negative condition is mostly due to complete deletion of RHD gene. So RhD protein and D antigen are not formed.
In African descent population, Rh negative condition may occur due to RHD pseudogene. This gene has premature stop codon and stops normal RhD protein formation.
Rh negative may also occur due to hybrid RHD gene. This gene fails to produce proper D antigen on RBC membrane.

Rh positive and Rh negative blood types are important in blood transfusion. Rh negative person may form anti-D antibody after receiving Rh positive blood.

Rh status is also important in pregnancy. Rh negative mother with Rh positive fetus may form anti-D antibody. This antibody can destroy fetal red blood cells.

Inheritance of The Rh Blood Group System

The following are the inheritance of Rh blood group system–

Rh blood group inheritance is mainly controlled by two genes. These are RHD gene and RHCE gene. Both genes are present on chromosome 1.
RHD and RHCE genes are closely linked genes. They are present very near to each other. So they are usually inherited together.
When these genes are inherited together as one unit, it is called haplotype. One Rh haplotype is inherited from mother. Another Rh haplotype is inherited from father.
Rh(D) antigen is inherited as autosomal dominant character. It means only one functional RHD gene is enough to show D antigen on red blood cells.
If a person receive one functional RHD gene from either mother or father, then D antigen is expressed. So the person becomes Rh positive.
Rh positive blood may be homozygous or heterozygous. Homozygous means both inherited genes are functional for D antigen. Heterozygous means only one functional gene is present.
Rh negative blood type means absence of functional RHD gene. So D antigen is not formed on the surface of red blood cells.
If both parents are Rh negative, then they do not have functional RHD gene. So the child will be Rh negative.
But Rh negative child can also be born from two Rh positive parents. This happens when both parents carry one hidden non-functional RHD gene.
This non-functional form is often shown as small d in genetic writing. There is no actual d antigen. Small d only means absence of D antigen.
Example, if both parents are Dd, then they are Rh positive. But they can produce dd child. This dd child will be Rh negative.
RHCE gene is inherited along with RHD gene. It controls the formation of C, c, E and e antigens.
The alleles of RHCE gene are inherited in fixed combinations. These combinations may be Ce, ce, cE or CE.
These combinations are present on same chromosome with RHD or without RHD. So different Rh haplotypes are formed.
In old time, Rh inheritance was explained by different genetic theories. One was Fisher-Race theory. Another was Wiener theory.
Fisher-Race theory said that Rh system is controlled by three closely linked genes. These were D, C/c and E/e.
Wiener theory said that Rh system is controlled by single gene locus. This single gene was thought to produce many antigens.
Modern molecular genetics showed that Rh blood group system is controlled mainly by two genes. These are RHD and RHCE. So the real genetic condition is between these two old theories.

Mechanism of Rh Sensitization

The following are the step by step mechanism of Rh sensitization–

In the first condition, the person is Rh negative. The red blood cells of this person do not contain Rh(D) antigen. So D antigen is absent on the cell surface.
The immune system of Rh negative person does not recognize D antigen as own antigen. So when D antigen enters the body, it is treated as foreign antigen.
The Rh negative person is exposed to Rh positive red blood cells. This exposure may occur during pregnancy, delivery, miscarriage or blood transfusion.
During pregnancy, Rh positive fetal blood may mix with Rh negative mother blood. This mixing is called fetomaternal hemorrhage. It is common during childbirth.
Exposure may also occur due to incompatible blood transfusion. It may also occur due to invasive obstetric procedure or abdominal trauma.
After exposure, the immune system detects Rh(D) antigen present on foreign red blood cells. This starts primary immune response.
The antigen presenting cells process the D antigen. Then immune cells are activated against this foreign antigen. This is the beginning of sensitization.
The immune system starts producing anti-D antibodies. At first mainly IgM antibody is formed. IgM is large antibody and it does not cross placenta.
Later, antibody production changes into IgG anti-D antibody. IgG is smaller antibody. It can cross placenta.
Once anti-D antibodies are formed, the person becomes sensitized. This is also called alloimmunization. The immune system keeps memory against Rh(D) antigen.
These antibodies and memory cells remain for long time. So the sensitized person can respond quickly if Rh positive blood enters again.
During second exposure, the immune system recognizes D antigen rapidly. This may happen in next pregnancy with Rh positive fetus or later transfusion.
In this second exposure, a secondary immune response occurs. It is also called anamnestic response. Large amount of IgG anti-D antibodies are produced.
In pregnancy, these IgG anti-D antibodies cross the placenta. They enter fetal circulation. Then they bind with Rh positive fetal red blood cells.
The antibody coated fetal RBCs are destroyed by macrophages. Mainly destruction occurs in spleen. This is called extravascular hemolysis.
Due to destruction of fetal RBCs, fetal anemia occurs. Severe condition may cause hemolytic disease of fetus and newborn (HDFN).
In transfusion, same anti-D antibodies attack transfused Rh positive red blood cells. This causes delayed hemolytic transfusion reaction (HTR).
So Rh sensitization is the immune response of Rh negative person against Rh(D) antigen after exposure to Rh positive red blood cells.

Formation of Anti-Rh Antibodies

The following are the step by step process of anti-Rh antibody formation-

In the first condition, the person is Rh negative. The red blood cells of this person do not have Rh antigen. Most commonly D antigen is absent.
Due to absence of D antigen, the body does not recognize it as own antigen. So if Rh antigen enters the body, it is treated as foreign antigen.
The Rh negative person is exposed to Rh positive red blood cells. This may occur by mismatched blood transfusion. It may also occur during pregnancy and delivery.
During pregnancy, small amount of fetal blood may enter the maternal blood. This is called fetomaternal hemorrhage. It mostly occurs during delivery.
The fetal red blood cells may carry Rh(D) antigen. When these cells enter Rh negative mother blood, the immune system detects the foreign antigen.
Rh antigens are highly immunogenic. They can easily stimulate immune response. So the immune system starts reaction against Rh positive red blood cells.
The foreign Rh antigen is processed by immune cells. Then lymphocytes are activated. This starts the primary immune response.
The immune system begins to form antibodies against Rh antigen. These antibodies are called anti-Rh antibodies. Most important is anti-D antibody.
This first antibody formation is called alloimmunization. It is also called sensitization. The person now becomes sensitized to Rh factor.
In early response, some IgM antibodies may be formed. IgM is large antibody. It usually cannot cross placenta.
Later most anti-Rh antibodies are formed as IgG antibody. IgG anti-D antibody is clinically important. It can cross placenta easily.
Once anti-Rh antibodies are formed, they do not disappear completely. The body keeps immunological memory against Rh antigen.
Memory cells remain in the body for long time. So the person becomes permanently sensitized. Next exposure gives faster reaction.
If the person is exposed again to Rh positive blood, secondary immune response occurs. This may happen in next pregnancy or another transfusion.
In secondary response, large amount of IgG anti-D antibodies are formed quickly. These antibodies bind with Rh positive red blood cells.
The antibody coated red blood cells are marked for destruction. Macrophages, mainly in spleen, destroy these cells. This is called extravascular hemolysis.
In pregnancy, IgG anti-D antibodies cross placenta and attack fetal Rh positive RBCs. This causes hemolytic disease of newborn (HDN) or HDFN.
In transfusion recipient, the same antibody reaction destroys transfused Rh positive red blood cells. This causes delayed hemolytic transfusion reaction.

Rh Incompatibility

Rh incompatibility is a condition where Rh negative blood mixes with Rh positive blood. It mainly occurs when Rh negative mother carries Rh positive baby.

Rh incompatibility occurs due to difference in Rh(D) antigen. Rh negative person does not have D antigen on red blood cells. Rh positive person has D antigen on red blood cells.
When Rh positive red blood cells enter into Rh negative person, the immune system identifies D antigen as foreign antigen. Then immune reaction may start.
This exposure may occur through mismatched blood transfusion. It may also occur during pregnancy or delivery when fetal blood enters mother blood.
In pregnancy, Rh negative mother and Rh positive fetus may create this condition. Small amount of fetal Rh positive blood can enter maternal circulation.
After exposure, mother immune system starts producing anti-D antibodies. This process is called Rh sensitization or alloimmunization.
First pregnancy is usually less affected. Because antibody formation takes some time. But antibodies remain in mother body after sensitization.
In next pregnancy, if fetus is again Rh positive, these anti-D antibodies can act quickly. They cross the placenta and enter fetal blood.
The maternal anti-D antibodies bind with fetal Rh positive red blood cells. Then fetal RBCs are destroyed. This causes hemolytic disease of fetus and newborn (HDFN).
HDFN is also called Rh disease. It may cause fetal anemia, jaundice and severe complication in newborn.
In blood transfusion, Rh incompatibility is also important. If sensitized Rh negative person receives Rh positive blood, then anti-D antibodies attack transfused RBCs.
This produces hemolytic transfusion reaction. In this condition transfused red cells are destroyed inside the body.
Rh incompatibility can be prevented in pregnancy by Rh immune globulin (RhIG). RhoGAM is one example of RhIG.
RhIG is given to Rh negative mother. It prevents formation of dangerous anti-D antibodies. So it helps to protect Rh positive fetus in future pregnancy.

Rh Blood Group Typing and Testing

Rh blood group typing is a laboratory test used to detect Rh(D) antigen on the surface of red blood cells. It tells whether blood is Rh positive or Rh negative.

Basic Rh(D) typing

Rh typing is generally done by serological method. It is similar to ABO forward grouping. Here patient red blood cells are tested with anti-D reagent.
Blood sample is collected in EDTA tube. Usually 0.5 to 4 ml blood may be used. EDTA prevents clotting and preserves the red blood cells.
In this test, patient RBCs are mixed with reagent containing anti-D antibodies. Then the mixture is observed for agglutination.
If RBCs clump after mixing with anti-D reagent, then D antigen is present. The blood is reported as Rh positive.
If no clumping occurs with anti-D reagent, then D antigen is absent or not detected. The blood is generally reported as Rh negative.

Weak and partial D testing

Sometimes RBCs show no agglutination or very weak agglutination with anti-D reagent. In this condition further testing is needed.
Antihuman globulin (AHG) test is used to detect weak reaction. AHG reagent enhances the reaction which is not clearly visible in routine typing.
In weak D phenotype, first reaction may be weak or absent. But after adding AHG reagent, moderate or strong agglutination may occur.
Weak D means D antigen is present but expressed in low amount on RBC membrane. So it may not be detected properly by routine method.
Partial D means some parts of D antigen are absent. This person may form anti-D antibody against the missing part of antigen.

Molecular genotyping

Molecular genotyping is DNA based testing. It is used when serological test cannot clearly identify the Rh type.
This test identifies the exact RHD allele present in the person. So it helps to differentiate weak D, partial D and other Rh variants.
Weak D type 1, 2 and 3 are usually treated as Rh positive. These persons can receive Rh positive blood and usually do not need RhIG during pregnancy.
Partial D persons are treated as Rh negative in important clinical condition. They should receive Rh negative blood and RhIG may be needed in pregnancy.
Genotyping helps to avoid unnecessary use of Rh immune globulin (RhIG). It also helps to save Rh negative red cell units.
PCR based genotyping can also be used for father testing. It checks whether father has one or two copies of Rh positive gene.
This is called paternal zygosity testing. It helps to know the risk of Rh disease in unborn baby.

Direct antiglobulin test

Direct antiglobulin test (DAT) is also called direct Coombs test. It does not detect antigen like routine Rh typing.
DAT detects whether antibodies are already attached to red blood cells inside the body. So it shows internal sensitization of RBCs.
This test is useful when antibody mediated RBC destruction is suspected. It is done in case of hemolytic transfusion reaction.
DAT is also important in hemolytic disease of fetus and newborn (HDFN). It helps to detect antibody coated fetal or newborn red blood cells.

Clinical Significance of The Rh Blood Group System

The following are the clinical significance of Rh blood group system–

Rh blood group system is very important in blood transfusion. It is the second important blood group after ABO blood group. Antibodies like anti-D, anti-C, anti-c, anti-E and anti-e may destroy incompatible transfused red blood cells and produce hemolytic transfusion reaction (HTR).
Hemolytic transfusion reaction due to Rh antibodies is mostly delayed type. The destruction of red blood cells occurs mainly outside the blood vessels. This is called extravascular hemolysis.
Rh blood group system is a major cause of hemolytic disease of fetus and newborn (HDFN). D antigen is most important in this condition. It causes about 50% of maternal alloimmunization.
HDFN occurs when Rh negative mother is exposed to Rh positive fetal blood. Mother forms IgG anti-D antibody. These antibodies cross placenta and destroy fetal red blood cells in present or future pregnancy.
Rh immune globulin (RhIG) is used in pregnancy as preventive treatment. It is given to Rh negative mother. It prevents mother immune system from forming harmful anti-D antibodies. RhoGAM is one example.
Chronic transfusion patients have high risk of alloimmunization. It is commonly seen in sickle cell disease (SCD) patients. They need repeated transfusion and so antibody formation may occur.
Many SCD patients have R₀ (Dce) phenotype. This phenotype is common in African descent population but rare in Caucasian donor pool. So finding matched blood may be difficult.
In chronic transfusion, blood should be matched for Rh antigens like C, E, c and e. It should also be matched for Kell antigen. This helps to prevent antibody formation.
Rh-null phenotype is also clinically important. It is also called golden blood. In this condition all Rh antigens are absent from red blood cells.
In Rh-null phenotype, RBC membrane structure becomes abnormal. It causes mild hemolytic anemia. This condition is called Rh deficiency syndrome.
Rh-null persons have serious transfusion problem. They can form antibodies against normal Rh-bearing blood. So they can receive blood only from another rare Rh-null donor.
RHD genotyping is used to identify unclear Weak D blood types. It helps to separate Weak D from Partial D when serological test is not clear.
Weak D type 1, 2 and 3 can be treated as Rh positive. These persons usually do not need RhIG. This helps to reduce unnecessary RhIG use and save Rh negative blood units.
Rh phenotype may also be related with susceptibility to some infections. Some studies shows relation with latent toxoplasmosis, non-SARS-CoV-2 and SARS-CoV-2 infection. This is mainly research based point.

Complications Associated with Rh Incompatibility

The following are the complications associated with Rh incompatibility–

Anemia is a common complication of Rh incompatibility. It occurs due to destruction of fetal red blood cells by maternal anti-D antibodies. The hemoglobin level becomes low and the fetus or newborn becomes pale.
Jaundice occurs due to rapid breakdown of red blood cells. During this process large amount of unconjugated bilirubin is formed. The skin, eyes and mucous membrane of newborn becomes yellow.
Kernicterus is a serious complication of severe jaundice. It occurs when excess bilirubin enter the brain. It may cause permanent brain damage in newborn.
Hepatosplenomegaly occurs when liver and spleen become enlarged. These organs try to produce more blood cells. This is done to compensate the anemia.
Hydrops fetalis is a severe condition. It occurs due to extreme anemia and heart failure. Fluid collects in fetal tissues and body cavities. There may be generalized edema, pleural effusion and ascites.
Dyspnea may occur in newborn. It is due to low oxygen supply and fluid collection in lungs. The newborn shows respiratory distress and difficulty in breathing.
Preterm birth may occur in severe Rh incompatibility. The baby may be delivered before normal time. Such newborn may need NICU care.
Fetal or neonatal death may occur in untreated severe condition. It may occur due to severe anemia, heart failure and massive destruction of fetal RBCs. Death may occur inside uterus or after birth.
Massive hemolysis may occur in Rh incompatible blood transfusion. The immune system attacks transfused red blood cells. Large amount of hemoglobin is released into the blood.
Shock and organ failure may occur after severe transfusion reaction. The body shows strong immune reaction against mismatched blood. It may cause systemic shock and acute kidney failure.
Disseminated intravascular coagulation (DIC) may occur in severe transfusion reaction. Clotting occurs inside blood vessels. At same time severe bleeding may also occur.

References

ABO and Rh blood groups and risk of infection: Systematic review and meta-analysis. (n.d.). PubMed Central (PMC).
Anti-G evaluations in D– pregnant women determine the need for Rh immune globulin prophylaxis: Report of two illustrative cases. (n.d.). ResearchGate.
Anti-G with concomitant anti-C and anti-D: A case report in a… (n.d.). PubMed Central (PMC).
Antibody information. (n.d.). LifeServe Blood Center.
Antibody-mediated antigen loss switches augmented immunity to antibody-mediated immunosuppression. (n.d.). PubMed Central (PMC).
Antibody-mediated immune suppression by antigen modulation is… (n.d.). PubMed Central (PMC).
Antibody-mediated immunosuppression can result from RBC antigen loss independent of Fcγ receptors in mice. (n.d.). PubMed Central (PMC).
Baronas, J., Westhoff, C. M., Vege, S., Mah, H., Aguad, M., et al. (2016). RHD zygosity determination from whole genome sequencing data. Journal of Blood Disorders & Transfusion, 7, 365. https://doi.org/10.4172/2155-9864.1000365
Bergström, J. J. E., Xu, H., & Heyman, B. (2017). Epitope-specific suppression of IgG responses by passively administered specific IgG: Evidence of epitope masking. Frontiers in Immunology, 8, 238. https://doi.org/10.3389/fimmu.2017.00238
Blood groups systems. (n.d.). PubMed Central (PMC) – NIH.
Chaffin, J. (n.d.). f Antigen. Blood Bank Guy Glossary.
Chaffin, J. (n.d.). Glossary: Weak D phenotype. Blood Bank Guy.
Chen, Q. (2022). (ISBT 004) RHD blood group alleles v6.2 30-SEP-2022. International Society of Blood Transfusion (ISBT).
Dean, L. (2005). Blood groups and red cell antigens. National Center for Biotechnology Information (US). https://www.ncbi.nlm.nih.gov/books/NBK2261/
Dean, L. (2005). Blood groups and red cell antigens. National Center for Biotechnology Information (US). University of California Los Angeles Library.
Dean, L. (2005). The Rh blood group. In Blood groups and red cell antigens. National Center for Biotechnology Information (US). https://www.ncbi.nlm.nih.gov/books/NBK2269/
Figure 1. Schematic structure of the RH gene locus. (A) The positions… (n.d.). ResearchGate.
Floch, A., & Westhoff, C. M. (2022). (ISBT 004) RHCE blood group alleles v6.2-31-MAR-2022. International Society of Blood Transfusion (ISBT).
Identification of rare anti-f alloantibody in a tertiary care center in… (n.d.). PubMed Central (PMC).
Jaladi, S., Cao, W., Meinecke, D. R., Ruegsegger, P. A., Weiss, J., Rhoades, J., & Connor, J. (2024). Anti-G evaluations in D- pregnant women determine the need for Rh immune globulin prophylaxis: Report of two illustrative cases. Immunohematology, 40(4), 145-148. https://doi.org/10.2478/immunohematology-2024-019
Jator, E. K., & Pedde, E. (2010). Anti-f in a 24-year-old male: A case study. Clinical Laboratory Science, 23(2), 68-71.
Keller, M. A. (2022, March 27). How to investigate those pesky serologic weak D samples. Red Cross Plus Online Blog. American Red Cross.
Maier, C. L., Mener, A., Patel, S. R., Jajosky, R. P., Bennett, A. L., Arthur, C. M., Hendrickson, J. E., & Stowell, S. R. (2018). Antibody-mediated immune suppression by antigen modulation is antigen-specific. Blood Advances, 2(21), 2986-3000. https://doi.org/10.1182/bloodadvances.2018018408
Partial D, weak D, and RhD genotyping: When to go beyond serology. (n.d.). Grifols Diagnostic Solutions Inc.
Review: The molecular basis of the Rh blood group phenotypes. (n.d.). PubMed Central (PMC).
Rh blood group system. (2026, June 15). In Wikipedia. https://en.wikipedia.org/wiki/Rh_blood_group_system
Rh immune globulin (RhIG). (2025). Allo Hope Foundation.
Rh-null vs. Ro blood type: What is the difference? (n.d.). E & J Bio UK Ltd.
Rhees, J. R. (n.d.). Introduction to the Rh blood group. University of Utah Department of Pathology, ARUP Laboratories.
Rho(D) immune globulin. (2026, May 27). In Wikipedia. https://en.wikipedia.org/wiki/Rho(D)_immune_globulin
Rosenkrans, D., Zubair, M., & Doyal, A. (2023). Rh blood group system. In StatPearls. StatPearls Publishing. https://www.ncbi.nlm.nih.gov/books/NBK594252/
Sandler, S. G., Flegel, W. A., Westhoff, C. M., Denomme, G. A., Delaney, M., Keller, M. A., Johnson, S. T., Katz, L., Queenan, J. T., Vassallo, R. R., & Simon, C. D. (2015). Joint statement on phasing-in RHD genotyping for pregnant women and other females of childbearing potential with a serologic weak D phenotype background. Association for the Advancement of Blood & Biotherapies (AABB).
Serological weak D phenotypes: A review and guidance for… (n.d.). PubMed Central (PMC).
Smith, Y. (2022, December 28). What is Rh blood group? News-Medical.Net.
The RH system. (n.d.). NHS Blood Donation.
The Rh blood group system: Comprehensive analysis of genetics, biochemistry, clinical significance, and immunoprophylaxis. (n.d.).
The genetics of the Rhesus blood group system. (n.d.). PubMed Central (PMC).
The structure and function of the Rh antigen complex. (n.d.). PubMed Central (PMC).
When you need Rh immune globulin. (n.d.). MyHealth Alberta.

Rh Blood Group System – Definition, Antigens, Antibody