Red Cell Alloimmunization (Isoimmunization)

last authored: April 2010, David LaPierre
last reviewed: August 2010, James Andrews, MD





Red cell alloimmunization (also called isoimmunization) is the production of antibodies targeted against red blood cells.  This occurs in an individual when they are exposed to foreign red cell antigens.  There are over 400 red cell antigens that have been identified. 

If a sensitized pregnant woman has red cell antibodies directed against a specific antigen present on the fetal red cells, several obstetrical complications may occur from transmission of these antibodies across the placenta to the fetus.  This may result in fetal anemai, hyperbilirubinemia, hydrops fetalis and possobly fetal death.

Several different groups of red cell antigens have been described:

ABO Blood Groups

ABO incompatibility is the most common cause of hemolytic disease of the newborn.  However, the anemia that results is usually mild.  Approximately 5 percent of cases will be clinically important.

CDE or Rhesus Blood Groups
There are five red cell antigens in this grouping: c (little c), C (big C), D, e (little e) and E (big E).  (There is no little D).  An Rh negative blood group refers to the absence of the D-antigen on the red blood cell (can also be called D-negative).  These groups are clinically important since many Rh(D)-negative individuals will become sensitized when exposed to Rh(D)-positive blood.  The c, C, e, E antibodies are not as immunogenic as the D-antigen, however they are all capable of causing fetal anemia.  As a result, all pregnant women should screened for the presence or absence of the D-antigen on their red cells and for anti-red cell antibodies in their serum.

The incidence of CDE antigens varies according to ethnicity.  Approximately 85% of caucasions are Rh(D)-positive whereas 92% of African-Americans and almost 99% of Native Americans and Asians are Rh(D)-positive.  The Basques have the highest incidence of Rh(D)-negativity with a rate of 34%. 

Other Red Cell Antigens
There are many other abnormal anti-red cell antibodies that have been associated with fetal anemia and hydrops fetalis (called minor red cell antigens).  Antibodies directed against the Kell (K) antigen, Duffy (Fy), Kidd (Jk) have all been associated with severe fetal anemia and hydrops fetalis.

Prevention of Rh alloimmunization by the administration of anti-D immune globulin (WinRHO, RhoGAM) has effectively decreased the number of cases of Rh(D) alloimmunization.  Maternal alloimmunization occurs in approximately 0.4 per 1000 births or in 1 to 2 percent of Rh(D)-negative women in Canada.  The most common reason is usually from failure to provide immunoprophylaxis to eligible women.

An Rh(D)-negative mother is at risk of Rh(D) alloimmunization (sensitization) if the fetus is Rh(D)-positive.  Without immunopropylaxis, this would occur in 12-16% of pregnancies in Rh(D)-negative women.

The risk of alloimmunization of an Rh(D)-negative mother with an Rh(D)-postivie infant is 16% overall.  2% would occur antepartum, 7% would occur by 6 months postpartum and 7% would be 'sensibilized.'  Women who are sensibilized have anti-D antibodies produced at undetectable levels during or after the index pregnancy, but antibodies are identified early in a subsequent pregnancy.




The Case of Mabel Utingua

Mabel is a 34 year-old woman with pregnant with her second child when she develops vaginal bleeding ten weeks into her pregnancy. She knows her blood type is A negative. Why should she be concerned?

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Causes and Risk Factors

Maternal sensitization can occur following:

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In the majority of cases of red cell alloimmunization, fetal-maternal hemorrhage occurs antenatally or during delivery.  It also accepted that on occasion, intact cells or free DNA from the fetus may cross the placental barrier into the maternal circulation during the course of the pregnancy.  If the fetus is Rh(D)-positive and the mother is Rh(D)-negative, then allimmunization may occure resulting in maternal anti-D antibody production.  These IgG antibodies are able to cross the placenta and those directed at fetal red cell antigens can cause their destruction.  Alloiimmunization by other antigens occurs by a similar process.  Fetal red cells that are coated with anti-red cell antibodies are sequestered in the fetal spleen and liver where they are destroyed, resulting in fetal anemia.

If there is an ABO incompatibility betweem the mother and the fetus, then anti-A or anti-B antibodies will lyse the fetal red cell in the maternal circulation and destroy it along with the Rh(D) antigen before it can stimulate a maternal immune response.  In this scenario, alloimmunization is therefore less likely to occur.  

Fetal anemia results in several physiologic changes.  Erythroblasts are released from the fetal liver and fetal cardiac output increases.  As anemia progresses, tissue hypoxia develops.  Hydrops fetalis is a late finding in severe cases of fetal anemia and is defined as the accumulation of extracellular fluid in at least 2 different body compartments (e.g. ascites, pericardial effusion, hydrothroax, skin edema etc.)

week gestation






blood volume (ml)







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Signs and Symptoms

  • history
  • physical exam


Inquire into past obstetrical history, including:

  • deliveries
  • miscarriages or ectopic pregnacies
  • previous blood transfusions
  • previous administration of anti-D immune globulin

Physical Exam


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  • lab investigations
  • diagnostic imaging

Lab Investigations

Routine screening should be done at the first prenatal visit to determine blood group, Rh status, and the presence of maternal antibodies. If antibodies are detected, then the titre should be determined and should be measured serially throughout the pregnancy to monitor for increasing antibody production.  In general, titres <1:16 are considered benign, while >1:16 increases the risk for the development of fetal anemia.  There are some antibodies, such as anti-Kell that can cause fetal anemia with low titres. 


Blood type and screen


If the mother is Rh(D) negative, the father can be offered testing for blood typing if paternity is certain.  If the father is Rh(D)-negative, then fetus will be Rh(D) negative as well.  If the fatheir is Rh(D) positive, then the chance the fetus will be Rh(D) positive would depend on the genotype of the father (e.g. heterozygous or homozygous).  If anti-D antibodies are present in the mother, amniocentesis can be performed to determine fetal blood typing which can help direct the need for further surveillance in the pregnancy.

The Kliehauer-Betke (KB) test can be used to determine extent of fetal-maternal hemorrhage.  In this laboratory investigation, maternal blood is obtained and examined under the microscope after acid-elution.  Maternal cells are lysed and appear as ghost cells whereas fetal cells are stained.  The volume of fetal hemorrhage into the maternal circulation can then be calculated by knowing the percent of fetal cells present from the Kleihauer test using a formula:

Estimate of volume of fetal hemorrhage = (maternal blood volume) x (fetal cells in KB)

*Maternal blood volume is usually estimated to be 5000 ml

The above formula is used to determine the amount of Rh(D) immune globulin needed for appropriate prophylaxis (see below).

If maternal antibodies are present, then referral should be made to a maternal-fetal medicine specialist for assessment of fetal anemia.  Fetal ultrasound can be performed for to look for evidence of fetal hydrops.  There are several methods to assess for fetal anemia:

1. Cordocentesis to determine fetal red cell count and hematocrit.

2. Amniocentesis to look for bilirubin pigment (breakdown product from the destruction of red cells) in the amniotic fluid using spectral analysis.  The optical density at 450nm is then plotted on a Liley curve according to gestational age  to estimate the risk of fetal anemia and to plan further intervention. 

3. Middle cerebral artery (MCA) Doppler interogation (of the fetus).  With anemia, the blood is less viscous and therefore flows at a higher velocity.  The peak systolic velocity of the MCA in the fetus correlates well with the degree of fetal anemia (increased velocities associated with worsening anemia).  This method of surveillance for fetal anemia is in widespread use at present since it is non-invasive and therefore has minimal procedure related risk.

Diagnostic Imaging

A fetal ultrasound can show fetal hydrops, or total body edema.


Doppler ultrasound of baby's arteries can show flow velocity. The higher the velocity, the greater the likelihood of hemorrhage and anemia.

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Anit-D immune globulin (anti-Rh IgG) (RhoGam or WinRho) can be given to an Rh(D) negative woman to reduce the risk of alloimmunization.  Anti-D IgG is manufatcured from human plasma and therefore is a blood product and appropriate consent should be obtained before administration.  In Canada, donors are screened and steps are taken in processing to destroy virsuses.  As such, there has never been a documented case of infectious disease transmission with WinRho in Canada, however the potential remains.  Anit-D IgG is available in 2 doses of 120 and 300 micrograms.  In general, 12 micrograms of WinRho will cover 1 mL of fetal whole blood (24 micrograms will cover 1 mL of fetal red blood cells).

The mechanism of action of Anti-D IgG is not completely understood.  Theories include the rapid macrophage mediated destruction of anti-D coated fetal red cells or possible down regulation of antigen specific B cells before an immune response can occur.

Indications for administration:

1. 28 weeks gestation in a Rh(D) negative mother, 300 micrograms should be given routinely as prophylaxis (if given prior to 28 weeks, a repeat injection should be given 12 weeks later)

2. Postpartum - perform a Kleihauer-Betke test on the mother (see above) and administer appropriate dose of anti-D IgG depending on the amount of fetal-maternal hemorrhage that occured (12 micrograms covers 1 mL of fetal whole blood).  If the Kleihauer is negative and passive anti-D antibodies from previous adimistration of anti-D IgG are detected (suggesting that there is still enough WinRho circulating), then anti-D IgG may be withheld.  

3. Antepartum Bleeding - administer 120 micrograms anti-D IgG up to 12 weeks gestation and 300 micrograms thereafter.  A Kleihauer-Betke should be perfromed for bleeding in the second and third trimesters to determine the amount of fetal-maternal hemorrhage and to calculate the appropriate anti-D IgG dose to be given.  Repeat doses may be necessary for further episodes of bleeding.

4. Spontaneous or Induced Abortion - administer 120 micrograms if less than 12 weeks or 300 micrograms otherwise
5. Ectopic Pregnancy
6. Molar Pregnancy
7. Procedures (amniocentesis, chorionic villous sampling, cordocentesis) - obtain Kleihauer-Betke and administer 300 micrograms of anti-D IgG.  Further dosing may be required depending on the Kleihauer result.

8. External Cephalic Version - obtain Kleihauer and give minimum of 120 micrograms of anti-D IgG.  Further dosing may be required depending on the Kleihauer result.
9. Platelet Transfusion - platelets may contain a small amount of red blood cells which may be Rh(D) positive.  Treatment with 120 micorgams of anti-D IgG covers approximately 6 units of platelets.

Anti-D immune globulin should be administered within 72 hours of bleeding, delivery or performing a procedure where fetal-maternal hemorrhage may occur.  However it can be given up to 28 days after an event, but it may be less effective in preventing alloimmunization.

If alloimmunization has occured and the mother is producing anti-D antibodies, then there is no benefit to the administration of anti-D immune globulin.

Example using Kleihauer-Betke result to calculate required dose of anti-D IgG:
    Kleihauer-Betke result = 0.1% (0.001)
    Therefore volume of fetal whole blood in maternal circulation = 0.001 x 5000 mL = 5 mL
    5 mL of fetal whole blood x 12 micrograms of anti-D IgG (to cover each mL fetal blood) = 60 micrograms of anti-D IgG needed for prophylaxis


Treatment of an Rh(D) Alloimmunized Pregnancy

1. First Affected Pregnancy

Perform serial titres to look for increased antibody production and perform paternal genotyping if paternity is certain.  If the father is Rh(D) negative and paternity is certain, no further follow-up is necessary.  If the father is heterozygous for the D-antigen, amniocentesis can be offered after 15 weeks gestation to determine the fetal blood type (if the fetus is Rh(D) negative, then no further follow-up is necessary).  If the father is homozygous then the fetus will be Rh(D) positive and is at risk. 

When a critical antibody titre is reached (between 1:8 to 1:32) increased surveillance is warranted.  Serial fetal middle cerebral artery Dopplers should be performed every 1 to 2 weeks to assess for fetal anemia starting at 18 weeks gestation.  If Doppler is unavailable, then amniocentesis can be perforemed with spectral analysis of the fluid to look for bilirubin pigment (see above). 

If there is evidence of severe fetal anemia, then either delivery is indicated (depending on gestational age) or cordocentesis with intrauterine transfusion.

2. Second Affected Pregnancy

Management is the same as above, however titres are not as reliable and fetal anemia can occur without appreciating a significant rise in antibody titre.

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Consequences and Course

Anti-Rh(D) antibodies may result in fetal red blood cell destruction. In the neonate, the consequences range from mild hemolytic disease of the newborn (usually manifested as hyperbilirubinemia) to a severe life-threatening anemia. 

Consequences in the newborn include:

Management may include volume replacement, blood transfusion, exchange-transfusion, photo therapy and supportive care depending on the severity.

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Resources and References

Cunninghame et al.,  Isoimmunization.  Williams Obstetrics, 22nd Edition.  McGraw-Hill 2007.


Gabbe et al., Red Cell Alloimmunization.  Obstetrics, Normal and Problem Pregnancies, 5th Edition.  Mosby 2007.


Fung Kee Fung et al., Prevention of Rh Alloimmunization.  SOGC Clinical Practice Guidelines No. 133., September 2003.


Guidelines for Perinatal Antibody Screening and Rh(D) immune globulin administration, Rh Program of Nova Scotia and the Reproductive Care Program of Nova Scotia, March 2010.

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Topic Development

authors: David LaPierre

reviewers: James Andews


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