PATHOPHYSIOLOGY OF RH INCOMPATIBILITY

Rh incompatibility, also known as Rh disease, arises when an Rh-negative mother is exposed to Rh-positive fetal red blood cells. This exposure can occur through various mechanisms such as fetomaternal hemorrhage during pregnancy, trauma, invasive obstetric procedures, or normal delivery. The primary antigen involved in this condition is the D antigen of the Rh blood group system.

Mechanism of Sensitization

1. Initial Exposure: When an Rh-negative mother is first exposed to Rh-positive fetal blood cells, her immune system recognizes these cells as foreign and begins to produce antibodies against the Rh antigen. This process is known as sensitization.
2. Antibody Production: The maternal immune response generates immunoglobulin G (IgG) antibodies specific to the Rh antigen. These antibodies persist for life once produced.
3. Subsequent Pregnancies: In subsequent pregnancies with an Rh-positive fetus, these preformed IgG antibodies can cross the placenta into the fetal circulation. Here, they bind to the fetal red blood cells that express the Rh antigen.
4. Hemolysis: The binding of maternal antibodies to fetal red blood cells forms antigen-antibody complexes that lead to hemolysis (destruction) of these cells in the fetus. This results in a condition known as alloimmune-induced hemolytic anemia.

Factors Influencing Sensitization

1. Volume of Transplacental Hemorrhage: The amount of fetal blood entering the maternal circulation plays a significant role in sensitization.
2. Maternal Immune Response: The extent and efficiency of the mother’s immune response also determine whether sensitization occurs.
3. ABO Incompatibility: Concurrent ABO incompatibility between mother and fetus can reduce the risk of Rh sensitization because maternal antibodies against ABO antigens may destroy fetal red blood cells before significant sensitization to Rh antigens occurs.

Clinical Implications

1. First Pregnancy: Most firstborn infants with an Rh-positive blood type are not affected by severe hemolytic disease because it takes time for maternal antibody production to reach levels that can cause significant harm.
2. Subsequent Pregnancies: With each subsequent pregnancy involving an Rh-positive fetus, the risk and severity of hemolytic disease increase due to pre-existing maternal antibodies.
3. Severity of Disease: The severity ranges from mild anemia in second pregnancies to severe cases where infants may die in utero from massive antibody-induced hemolytic anemia.
4. Prevention and Treatment: Administration of RhoGAM (Rh immunoglobulin) during and after pregnancy can prevent sensitization by neutralizing any fetal red blood cells that enter maternal circulation before they trigger an immune response.

In summary, Rh incompatibility involves a complex interplay between initial exposure, antibody production, and subsequent immune responses that lead to hemolysis in fetuses with incompatible blood types, primarily affecting pregnancies beyond the first one unless preventive measures like RhoGAM are employed effectively.

5 Major Rh Antigens

The Rh blood group system is one of the most complex and clinically significant blood group systems in humans. It consists of over 50 antigens, but five of these are considered major due to their prevalence and clinical importance. These five major Rh antigens are D, C, c, E, and e.

1. D Antigen

The D antigen is the most significant antigen in the Rh system. Its presence or absence on red blood cells determines whether a person is Rh-positive (D+) or Rh-negative (D-). The D antigen is highly immunogenic, meaning it can provoke a strong immune response if transfused into someone who lacks it. This can lead to hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (HDFN).

2. C Antigen

The C antigen is another important component of the Rh system. It is encoded by the RHCE gene and can be found in combination with other antigens like D or E. The presence of the C antigen can influence compatibility in blood transfusions and organ transplants.

3. c Antigen

The c antigen is also encoded by the RHCE gene but represents a different allele than the C antigen. Like other Rh antigens, it plays a crucial role in determining compatibility for blood transfusions and can be involved in alloimmunization if mismatched.

4. E Antigen

The E antigen is one of the less common but still significant Rh antigens. It is also encoded by the RHCE gene and can exist alongside other antigens like D or C. The E antigen must be matched carefully during transfusions to prevent immune reactions.

5. e Antigen

The e antigen is one of the most frequently occurring Rh antigens after D. It is encoded by a different allele of the RHCE gene than E. Like other major Rh antigens, its presence or absence must be considered during blood typing for transfusions to avoid adverse reactions.

These five major antigens are critical for ensuring safe blood transfusions and managing conditions like HDFN effectively.

Characteristics of HDN

Hemolytic disease of the newborn (HDN), also known as erythroblastosis fetalis, is a condition that occurs when there is an incompatibility between the blood types of a mother and her fetus. This condition typically arises when a mother with Rh-negative blood type is carrying a fetus with Rh-positive blood type. The most common form of HDN is Rh incompatibility, but it can also occur due to other blood group antigens such as ABO or Kell.

One of the key characteristics of HDN is the production of maternal antibodies against fetal red blood cells. When there is an incompatibility between the blood types of the mother and fetus, the mother’s immune system may recognize the fetal red blood cells as foreign invaders and produce antibodies to attack them. These antibodies can cross the placenta and destroy the fetal red blood cells, leading to hemolysis (breakdown of red blood cells) and subsequent anemia in the fetus.

Another characteristic of HDN is jaundice in the newborn. As a result of hemolysis, the breakdown of red blood cells releases bilirubin, a yellow pigment that can accumulate in the baby’s bloodstream and tissues, causing jaundice. Severe jaundice can lead to complications such as kernicterus, a rare but serious condition characterized by brain damage due to high levels of bilirubin.

In severe cases of HDN, affected infants may exhibit symptoms such as pallor, lethargy, poor feeding, and enlarged liver and spleen. Without prompt diagnosis and treatment, HDN can result in serious complications including severe anemia, hydrops fetalis (excessive fluid accumulation in fetal tissues), and even death.

Treatment for HDN may involve interventions to manage jaundice such as phototherapy or exchange transfusion to replace the infant’s damaged red blood cells with compatible donor cells. In cases where severe anemia or hydrops fetalis has developed, intrauterine transfusions may be necessary to support the fetus until delivery.

Establishing the Diagnosis of HDN

1. Identifying Risk Factors: To diagnose Hemolytic Disease of the Newborn (HDN), it is essential first to identify risk factors that could indicate a potential incompatibility between the mother’s and baby’s blood types. The primary risk factor is an Rh-negative mother carrying an Rh-positive baby, especially if she has been sensitized in a previous pregnancy or due to other events like miscarriage, abortion, or invasive prenatal testing.

2. Prenatal Testing: During pregnancy, several tests can help establish the diagnosis of HDN:

• Blood Test: A maternal blood test can detect the presence of Rh antibodies. If these antibodies are found, it indicates that the mother has been sensitized and her immune system is prepared to attack Rh-positive red blood cells.
• Ultrasound: An ultrasound can reveal signs of fetal distress such as enlarged organs (liver, spleen, heart) or fluid accumulation in body cavities (hydrops fetalis). These signs suggest that the fetus may be suffering from anemia and other complications due to hemolysis.
• Amniocentesis: This procedure involves inserting a needle through the abdominal wall into the amniotic sac to collect a sample of amniotic fluid. The fluid is then tested for bilirubin levels, which can indicate the degree of hemolysis occurring in the fetus.
• Percutaneous Umbilical Cord Blood Sampling (PUBS): Also known as fetal blood sampling, this test involves taking a blood sample directly from the umbilical cord. The sample is analyzed for anemia, bilirubin levels, and the presence of maternal antibodies attacking fetal red blood cells.

3. Postnatal Testing: After birth, additional tests are conducted to confirm HDN:

• Physical Examination: A thorough physical examination of the newborn may reveal symptoms such as pale skin (anemia), jaundice (yellowing of skin and eyes), an enlarged liver and spleen, or generalized swelling (hydrops fetalis).
• Blood Tests: Newborns suspected of having HDN will undergo several blood tests:
  • Complete Blood Count (CBC): This test measures various components of blood including red blood cell count and hemoglobin levels to assess anemia.
  • Direct Coombs Test: This test detects antibodies attached to red blood cells in the newborn’s bloodstream.
  • Bilirubin Levels: Elevated bilirubin levels indicate excessive breakdown of red blood cells.

4. Differential Diagnosis: It is crucial to differentiate HDN from other conditions that might present with similar symptoms such as neonatal jaundice due to other causes or congenital infections. This ensures accurate diagnosis and appropriate treatment.

By systematically identifying risk factors, conducting prenatal and postnatal tests, and differentiating from other conditions with similar presentations, healthcare providers can establish a diagnosis of Hemolytic Disease of the Newborn effectively.

Differentiating Between ABO and Rh Hemolytic Disease of the Newborn (HDN)

ABO Hemolytic Disease of the Newborn (ABO HDN)

1. Cause:
   • ABO HDN occurs when there is an incompatibility between the mother’s and baby’s blood types within the ABO blood group system. This typically happens when a mother with blood type O has a baby with blood type A, B, or AB.
   • The mother’s immune system can produce antibodies against A or B antigens present on the baby’s red blood cells.
2. Incidence:
   • ABO HDN is more common than Rh HDN but usually less severe.
   • It can occur in first pregnancies because anti-A and anti-B antibodies are naturally occurring.
3. Severity:
   • The severity of ABO HDN is generally mild to moderate.
   • Symptoms may include mild anemia and jaundice, which are often treatable with phototherapy.
4. Diagnosis:
   • Diagnosis involves testing for maternal antibodies against fetal red blood cells, complete blood count tests for the baby, and monitoring bilirubin levels after birth.
5. Treatment:
   • Treatment usually includes phototherapy to manage jaundice and, in rare cases, exchange transfusions if bilirubin levels are extremely high.
6. Prevention:
   • There is no specific prevention for ABO HDN, but early detection and treatment can manage symptoms effectively.

Rh Hemolytic Disease of the Newborn (Rh HDN)

1. Cause:
   • Rh HDN occurs when an Rh-negative mother carries an Rh-positive baby.
   • The mother’s immune system produces antibodies against the Rh antigen on the baby’s red blood cells if she becomes sensitized during pregnancy or delivery.
2. Incidence:
   • Less common than ABO HDN due to preventive measures but potentially more severe.
   • Sensitization usually occurs during a previous pregnancy, miscarriage, abortion, or transfusion involving Rh-positive blood.
3. Severity:
   • The severity of Rh HDN ranges from mild to life-threatening.
   • Severe cases can lead to hydrops fetalis (severe swelling), severe anemia, hyperbilirubinemia, jaundice, enlarged liver/spleen, heart failure in utero, and kernicterus (brain damage).
4. Diagnosis:
   • Diagnosis involves maternal antibody screening during pregnancy, ultrasound monitoring for fetal anemia or hydrops fetalis, amniocentesis to measure bilirubin levels in amniotic fluid, and cordocentesis for direct fetal blood sampling.
5. Treatment:
   • Treatment during pregnancy may include intrauterine blood transfusions and early delivery if complications arise.
   • After birth, treatments include phototherapy for jaundice, intravenous fluids, oxygen therapy or mechanical ventilation if needed, exchange transfusions to replace damaged blood with fresh donor blood.
6. Prevention:
   • Prevention involves administering Rh immunoglobulin (Rho(D) immune globulin) to Rh-negative mothers during pregnancy and within 72 hours after delivery of an Rh-positive baby to prevent sensitization.

Fetomaternal Hemorrhage

Fetomaternal hemorrhage (FMH) is a condition characterized by the passage of fetal blood into the maternal circulation during pregnancy or childbirth. This can occur due to various reasons such as trauma, placental abruption, invasive procedures, or other complications during pregnancy. FMH can lead to significant risks for both the fetus and the mother, including fetal anemia, fetal distress, preterm birth, and maternal sensitization to fetal blood antigens.

This condition can occur in both normal and abnormal pregnancies, with varying degrees of clinical significance.

Normal Pregnancy

In a typical pregnancy, the maternal and fetal circulations are separated by a placental barrier composed of two layers: the syncytiotrophoblast and the cytotrophoblast. This barrier facilitates gas and nutrient exchange while preventing direct contact between maternal and fetal blood. However, it is estimated that less than 1ml of fetal blood may enter the maternal circulation during normal labor in approximately 96% of deliveries. Although this small amount is generally harmless, it can sometimes trigger an immune response in the mother, leading to conditions such as Rhesus disease of the newborn.

Abnormal Pregnancy

In cases where fetomaternal hemorrhage is more significant, it often results from trauma, placental abruption, or spontaneous events with no identifiable cause. Up to 30ml of fetal blood can be transferred to the maternal circulation without causing noticeable symptoms in either mother or fetus. However, larger volumes can lead to severe complications such as anemia in the fetus, which may result in neurologic injury, stillbirth, or neonatal death.

Treatment

Once FMH is diagnosed, management strategies depend on the severity of the hemorrhage and the gestational age of the fetus:

1. Emergency Caesarean Section: If there are signs of non-reassuring fetal status, an emergency C-section may be necessary to deliver the baby promptly.
2. Intravascular Transfusion: For fetuses diagnosed with severe anemia due to FMH, intravascular transfusion can be performed while still in utero. This procedure involves transfusing compatible red blood cells directly into the fetal circulation.
3. Postnatal Transfusion: In cases where FMH is identified after birth, neonatal transfusion may be required to stabilize the newborn.

Case Studies

Four cases from Mount Sinai Hospital illustrate these treatment options:

1. Three women presented with reduced fetal movements and underwent NSTs followed by Kleihauer-Betke tests and ultrasounds. All three required emergency C-sections due to non-reassuring fetal status.
2. One fetus received an intravascular transfusion before delivery.
3. Another case involved postnatal transfusion after spontaneous vaginal birth.
4. Neurological outcomes were favorable in all four cases.

Given its potential severity and underreporting issues, establishing a national registry could help improve understanding and management strategies for fetomaternal hemorrhage.

Tests Used for Detection of Fetomaternal Hemorrhage

1. Rosette Test

The Rosette Test is a qualitative screening method used to detect the presence of fetal red blood cells (RBCs) in the maternal circulation. This test is highly sensitive and can detect as little as 10 mL of fetal whole blood mixed with maternal blood. The procedure involves:

• Mixing maternal blood with anti-D antibodies.
• Incubating the mixture to allow anti-D antibodies to bind to any D-positive fetal RBCs.
• Adding indicator cells that form rosettes around the D-positive fetal cells, which are then counted under a microscope.

This test is primarily used for D-negative mothers carrying a potentially D-positive fetus. However, it cannot be used for D-positive mothers or when both mother and fetus are D-negative.

2. Kleihauer-Betke Acid-Elution Test

The Kleihauer-Betke Acid-Elution Test is a quantitative confirmatory test that measures the extent of fetomaternal hemorrhage by differentiating between fetal and adult hemoglobin (Hb). The steps include:

• Preparing a blood smear from the maternal sample.
• Treating the smear with an acid buffer, which elutes adult hemoglobin but leaves fetal hemoglobin intact.
• Staining the slide to visualize and count the remaining fetal RBCs under a microscope.

This test relies on the resistance of fetal hemoglobin (HbF) to acid-elution compared to adult hemoglobin, making it possible to quantify the number of fetal cells present in maternal blood. Despite its widespread use, this test lacks standardization and precision, especially in conditions with elevated F-cells.

3. Flow Cytometry

Flow Cytometry using anti-HbF antibodies is an advanced method for detecting and quantifying fetomaternal hemorrhage. The procedure involves:

• Labeling maternal blood samples with fluorescently tagged anti-HbF antibodies.
• Passing the labeled cells through a flow cytometer, which detects and counts HbF-containing cells based on fluorescence intensity.

Flow cytometry offers higher sensitivity and specificity compared to traditional methods like Kleihauer-Betke but requires specialized equipment and trained personnel, making it more costly.

4. Hematology Analyzers Adapted for Flow Cytometry

Some modern hematology analyzers have been adapted to perform flow cytometry-based detection of fetal cells. These automated systems offer an efficient alternative for clinical laboratories by integrating flow cytometric capabilities into routine hematological analysis.

These tests collectively help in accurately detecting and quantifying fetomaternal hemorrhage, thereby guiding appropriate clinical interventions such as Rh immune globulin administration to prevent Rh alloimmunization.

Blood Group to be Used in HDN

Hemolytic disease of the newborn (HDN), also known as erythroblastosis fetalis, is a condition that occurs when there is an incompatibility between the blood types of a mother and her fetus. This incompatibility can lead to the mother’s immune system producing antibodies that attack the baby’s red blood cells, causing severe complications such as anemia, jaundice, and even death in severe cases.

In cases of HDN, it is crucial to determine the blood group of both the mother and the fetus to assess the risk of hemolysis and plan appropriate treatment. The most important blood group systems involved in HDN are the ABO system and the Rh system. The ABO system classifies blood into four main groups: A, B, AB, and O. In HDN, if a mother with blood type O gives birth to a baby with blood type A or B, there is a risk of hemolysis due to anti-A or anti-B antibodies present in the mother’s blood.

The Rh system categorizes blood into Rh-positive and Rh-negative groups based on the presence or absence of the Rh antigen. In cases where an Rh-negative mother carries an Rh-positive fetus, there is a risk of sensitization during pregnancy or childbirth. This sensitization can lead to the development of anti-Rh antibodies in the mother’s blood, which may cause HDN in subsequent pregnancies with Rh-positive babies.

To prevent HDN in cases of Rh incompatibility, it is essential to administer Rh immunoglobulin (RhIg) to Rh-negative mothers at specific times during pregnancy and after childbirth. This treatment helps prevent sensitization and reduces the risk of hemolytic disease in future pregnancies.

In conclusion, determining the blood group compatibility between a mother and her fetus is crucial in managing and preventing HDN. Understanding the potential risks associated with different blood group combinations allows healthcare providers to implement appropriate interventions to safeguard both maternal and fetal health.