Fetal maternal hemorrhages can occur during the first and second trimesters or at term during separation of the placenta.Clinically, it is often important to know the source and extent of bleeding. Fetal RBCs can be quantitated using the Kleihauer-Betke technique.
The quantitation of fetal RBCs is useful in the following clinical situations:
- to determine if a vaginal bleed during pregnancy is from mother or fetus.
- to determine the source of blood in amniotic fluid
- to calculate the dose of Rh immune globulin needed to prevent Rh sensitization in an Rh negative women with a positive fetal blood screen postpartum.
If a woman is known to be D-negative and not immunized to D antigen, a test for excessive fetal maternal hemorrhage should be performed to determine the dosage of Rh immune globulin.
The Kleihauer Betke test relies on the principle that red cells containing fetal hemoglobin (HbF) are less susceptible to acid elution than cells containing HbA. A thin smear of maternal blood is exposed to citric acid phosphate buffer (pH 3.2), which elutes hemoglobin from maternal red cells, resulting in pale ghost cells. Fetal red cells are resistant to acid and retain their hemoglobin. Consequently, they stain pink with erythrosin B dye. The smear is examined microscopically to determine the percentage of fetal red blood cells. A minimum of 2000 cells should be counted and the ratio of fetal to adult red cells is determined.
Results are reported as the percent of fetal RBCs present. The sensitivity of the method is approximately 0.5 mL of fetal blood in the maternal circulation. This corresponds to about 1 fetal cell per 50,000 maternal cells. The Kleihauer-Betke stain may occasionally underestimate the number of fetal RBCs present due to the fact that the fetus begins to synthesize hemoglobin A in the last trimester of pregnancy. Fetal cells, which had completed the switch to adult hemoglobin, would be counted as adult cells. False positive reactions may occur when maternal RBCs have increased levels of hemoglobin F such as occurs in various hemoglobinopathies including hereditary persistence of fetal hemoglobin, thalassemia, and sickle cell anemia.
This test involves a considerable amount of subjective interpretation. The quality of the stain must be very good so that red cells can be clearly distinguished from leukocytes. Several published studies and proficiency surveys have demonstrated that the precision and accuracy of this method are poor. Variation from laboratory to laboratory is 50% and the rate of fetal cell detection is only 90%.
The amount of fetal maternal hemorrhage is calculated by multiplying the percent fetal cells by 50. This calculation assumes that maternal blood volume is 5000 mL or 50 dL. This product is then divided by 30, which is the volume of fetal whole blood neutralized by a single vial of RhIg (300 ug dose).
Vials of RhIg = % fetal cells x 50/30
A 30 mL fetal maternal hemorrhage is equivalent to 12 fetal red cells per 2000 adult red cells or 0.6%.
Because of the imprecision of the Kleihauer-Betke test in determining the dose of RhIg, the AABB Technical Manual recommends rounding up when the decimal point is 0.5 or higher and rounding down when the decimal point is <0.5. In either case one extra vial of RhIg is added to ensure that sufficient RhIg is given to neutralize all D positive fetal red cells.
For example, if the percent of fetal hemorrhage is 2%, then the volume of fetal hemorrhage is 2% x 50 = 100 mL. Dividing 100 mL by 30 mL/vial yields 3.3 vials. This number is rounded down to 3 and 1 vial is added for insurance. The required dose is 4 vials.
Reference range is no fetal RBCs present.
Specimen requirement is one 7 mL lavender top (EDTA) tube of blood or one mL of amniotic fluid or vaginal blood.
References
Kim YA, Makar RS, Detection of fetomaternal hemorrhage, Am J Hematol, 2012; 87: 417-423.
Australian and New Zealand Society of Blood Transfusion, Guidelines for Laboratory Estimation of Fetomaternal Haemorrhage, 2nd edition, September 2021.
How to resolve AdBlock issue?