Understanding Fetal-Maternal Hemorrhage: The Kleihauer Betke Test
The Kleihauer Betke (KB) test, also known as the acid elution test, is a medical test used to determine the amount of fetal blood that has entered the maternal bloodstream. This test is typically performed in cases of maternal trauma, placenta previa, and other conditions that may cause bleeding during pregnancy. It is also used to calculate the dose of Rh immune globulin (RhIg) that is needed to prevent Rh sensitization in Rh-negative mothers.
What is Rh Sensitization?
Rh sensitization occurs when an Rh-negative mother is exposed to Rh-positive fetal blood during pregnancy or delivery. This exposure can cause the mother's immune system to produce antibodies that attack and destroy Rh-positive blood cells. This can lead to hemolytic disease of the newborn (HDN), which can cause anemia, jaundice, and other complications.
Rh sensitization can be prevented by administering RhIg to the mother after exposure to Rh-positive fetal blood. RhIg works by binding to and neutralizing any Rh-positive fetal cells that may have entered the mother's bloodstream, preventing the mother's immune system from producing antibodies.
Why is Kleihauer Betke Test Preformed?
The Kleihauer Betke test is a medical test used to determine the amount of fetal red blood cells in the mother's bloodstream. This test is performed on pregnant women who have experienced bleeding during pregnancy or have a high risk of fetal-maternal hemorrhage due to trauma, placental abruption, or other conditions.
The test is particularly important for Rh-negative mothers who carry an Rh-positive fetus. If fetal-maternal hemorrhage occurs, the mother's immune system can develop antibodies against the Rh factor, which can lead to hemolytic disease of the newborn (HDN) in subsequent pregnancies. HDN can cause severe anemia, jaundice, and other complications in the newborn.
The Kleihauer Betke test allows healthcare providers to determine the amount of fetal blood that has entered the mother's bloodstream and to calculate the appropriate dose of Rh immune globulin (RhoGAM) to prevent the mother's immune system from attacking the Rh-positive fetus. This can help to prevent HDN and other complications in subsequent pregnancies.
Principles of the Kleihauer-Betke Test
The Kleihauer-Betke test takes advantage of the differences in the composition of fetal and adult hemoglobin to distinguish fetal RBCs from maternal RBCs in maternal blood. Fetal red blood cells contain fetal hemoglobin (HbF), which is more resistant to acid elution than adult hemoglobin (HbA). When a blood smear is treated with an acid solution, maternal RBCs (which contain HbA) will lose their hemoglobin and appear as pale "ghost cells," while fetal RBCs (containing HbF) retain their hemoglobin and remain stained red. This allows for the visualization and quantification of fetal RBCs in a maternal blood sample.
How is the Kleihauer Betke Test Performed?
The Kleihauer-Betke test is a relatively simple laboratory procedure that involves several key steps:
A. Blood Sample Collection
A sample of maternal blood is collected, typically from the mother’s arm. This sample is then prepared for analysis in the laboratory.
B. Slide Preparation
A blood smear is made by spreading a thin layer of maternal blood on a microscope slide. The smear is allowed to dry, and then it undergoes staining and acid treatment.
C. Acid Elution
The blood smear is treated with an acid solution. The acid selectively elutes (removes) the hemoglobin from maternal RBCs, which contain adult hemoglobin (HbA). Fetal RBCs, which contain fetal hemoglobin (HbF), are resistant to this process and retain their hemoglobin.
D. Staining
After acid elution, the slide is stained with a special dye, typically a counterstain such as eosin, which colors the cells. The fetal RBCs appear pink or red under the microscope, while the adult RBCs become pale or colorless (ghost cells).
E. Microscopic Examination
The stained slide is examined under a microscope. The number of fetal RBCs is counted in proportion to the total number of maternal RBCs present. The results are typically expressed as a percentage, representing the proportion of fetal RBCs among the total RBCs.
F. Calculation of Fetomaternal Hemorrhage Volume
Based on the proportion of fetal cells in the maternal blood, the volume of fetal-maternal hemorrhage can be estimated. This is important for determining the appropriate treatment, especially the dose of Rh immunoglobulin to administer to Rh-negative mothers.
Applications of the Kleihauer-Betke Test
The Kleihauer-Betke test has several important clinical applications, particularly in obstetric and perinatal care. Its primary role is to assess fetomaternal hemorrhage (FMH), but it is also used in trauma cases and for Rh incompatibility management.
A. Assessment of Fetomaternal Hemorrhage (FMH)
Fetomaternal hemorrhage occurs when fetal blood enters the maternal circulation. This can happen during childbirth, after trauma (e.g., car accidents or falls), or as a result of placental complications such as placental abruption. The Kleihauer-Betke test helps quantify the amount of fetal blood in the maternal circulation, which is important for managing the consequences of FMH, including fetal anemia or maternal alloimmunization.
B. Management of Rh Incompatibility
Rh incompatibility occurs when an Rh-negative mother carries an Rh-positive fetus. If fetal RBCs enter the maternal bloodstream, the mother’s immune system may produce antibodies against the Rh antigen, leading to hemolytic disease of the fetus or newborn in future pregnancies. The Kleihauer-Betke test helps determine the extent of fetomaternal hemorrhage and guides the administration of Rh immunoglobulin (RhIg), which prevents the mother's immune system from producing these harmful antibodies.
In cases of excessive fetomaternal hemorrhage, a higher dose of RhIg may be required to neutralize the Rh-positive fetal cells in the mother’s blood.
C. Trauma in Pregnancy
Pregnant women who experience trauma, such as car accidents or falls, are at risk of FMH. In these cases, the Kleihauer-Betke test can be used to assess whether fetal blood has entered the maternal circulation, helping clinicians evaluate the risk to the fetus and determine the need for interventions such as RhIg administration or close fetal monitoring.
D. Unexplained Fetal Distress or Stillbirth
In cases where fetal distress or stillbirth occurs, the Kleihauer-Betke test may be used to determine whether a large fetomaternal hemorrhage contributed to the adverse outcome. A significant presence of fetal RBCs in the maternal circulation may suggest that FMH played a role in the event.
Interpretation of Kleihauer-Betke Test Results
The results of the Kleihauer-Betke test are reported as the percentage of fetal red blood cells present in the maternal blood sample. Based on this percentage, the volume of fetomaternal hemorrhage can be estimated. For example:
Normal Results: In a normal pregnancy, a small amount of fetal blood may naturally pass into the maternal circulation. A very low percentage of fetal cells (e.g., less than 0.1%) may be detected.
Increased Fetal Cells: If the test reveals a significant number of fetal cells (e.g., greater than 0.1%), this indicates a more substantial fetomaternal hemorrhage. The estimated volume of hemorrhage is calculated to guide treatment, especially in determining the appropriate dose of Rh immunoglobulin.
The typical formula used for estimating the volume of fetal hemorrhage is based on the assumption that maternal blood volume is approximately 5000 mL, and each milliliter of maternal blood contains about 1,000,000 RBCs. The percentage of fetal cells can be used to estimate the total volume of fetal blood in maternal circulation.
Limitations and Considerations
While the Kleihauer-Betke test is a valuable tool, it has several limitations that may affect its accuracy and interpretation:
A. Subjectivity in Counting
The test relies on visual inspection under a microscope, and the process of counting fetal cells is somewhat subjective. Variability in counting techniques between different laboratory technicians can lead to discrepancies in results.
B. Limited Sensitivity
The Kleihauer-Betke test may not detect very small amounts of fetal blood in the maternal circulation. In cases of minor fetomaternal hemorrhage, the test may yield a false negative result.
C. Other Hemoglobin Variants
The test assumes that fetal cells contain only fetal hemoglobin (HbF) and that maternal cells contain adult hemoglobin (HbA). However, in some cases, adults may have elevated levels of HbF, such as in hereditary conditions like sickle cell anemia or thalassemia. This can lead to a false-positive result, where maternal cells are mistaken for fetal cells.
D. Alternative Methods
In some cases, flow cytometry, a more advanced laboratory technique, may be used as an alternative to the Kleihauer-Betke test. Flow cytometry can more accurately distinguish fetal cells based on specific surface markers (e.g., Rh antigen or HbF levels), providing more precise quantification of fetal RBCs in maternal blood.
Clinical Significance of the Kleihauer-Betke Test
The Kleihauer-Betke test plays an important role in obstetric care and the management of fetomaternal hemorrhage. Its primary significance lies in:
1.) Preventing Maternal Alloimmunization: By detecting fetal RBCs in the maternal circulation, the test helps prevent Rh alloimmunization in Rh-negative mothers, a critical step in protecting future pregnancies from hemolytic disease of the newborn.
2.) Managing Obstetric Complications: The test is valuable in evaluating potential fetal trauma following maternal injury or placental complications, allowing for timely interventions to protect both the mother and fetus.
3.) Assessing Fetal Well-being: In cases of unexplained fetal distress or stillbirth, the Kleihauer-Betke test can provide insights into whether fetomaternal hemorrhage was a contributing factor.
Summary
The Kleihauer-Betke test is a crucial diagnostic tool in obstetrics, providing valuable information about the presence and extent of fetal red blood cells in maternal circulation. It plays an essential role in managing Rh incompatibility, assessing fetomaternal hemorrhage after trauma, and helping to prevent complications that could affect both the mother and fetus.
