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Fetal Maternal Hemorrhage

A test for fetal maternal hemorrhage (FMH) should be performed approximately one hour after delivery on a maternal sample from all D negative women who deliver a D positive fetus. Testing for FMH should be done regardless of the presence of detectable passive anti-D in maternal serum.

The rosette test is a sensitive qualitative screening method that can detect 10 mL or more of fetal whole blood in the maternal circulation. A maternal blood sample is incubated with anti-D antibody which binds to any D positive fetal RBCs present in the suspension.  Maternal red cells are then washed to remove unbound antibody.  

A red cell suspension from a D negative mother is first incubated with monoclonal IgM anti-D for 5 minutes at room temperature and then washed to remove all unbound antibody. A dilute suspension of D positive indicator cells is then added, and the mixture of maternal red cells and indicator red cells is centrifuged. Since any minor population of D positive fetal red cells will have been coated with anti-D, the D positive indicator red cells will bind to them and form visible agglutinates (rosettes) around them.

After centrifugation, the mixture is resuspended and spread on a microscope slide. Five fields are examined at low power. Observation of four or fewer rosettes is considered negative. A few rosettes can be seen with a FMH of as little as 2.5 mL. If five or more rosettes are observed, the test is positive for D-positive fetal red cells. The presence of 5 or more rosettes indicates that a FMH of at least 30 mL has occurred. Approximately 0.3% of term deliveries have FMH of this magnitude.

The rosette test cannot detect FMH if the mother is D positive or the infant is D negative. The rosette test may be falsely positive if the mother has a variant of the D antigen known as weak D, and falsely negative if the fetus/neonate is weak D. A false positive result may also occur if the mother has a positive direct antiglobulin test (DAT) due to an autoantibody because of crosslinking and agglutination of the mother’s antibody coated red cells.

If the rosette test is positive, the degree of FMH should be quantified by using the Kleihauer Betke acid elution method or flow cytometry. 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%.

An alternative to the Kleihauer Betke test for quantitation of fetal cells is flow cytometry. The flow cytometric method utilizes a fluorescently labeled monoclonal antibody to the gamma chain of the HbF molecule (anti-HbF).  A sample of whole blood is fixed with glutaraldehyde to crosslink hemoglobin inside the cells and then cell membranes are permeabilized with a detergent to ensure access and binding of anti-HbF.  A flow cytometer determines the percentage of fetal cells by analyzing approximately 65,000 cells. Fetal red cells are clearly distinguished from adult cells by their significantly higher fluorescent signal. Proficiency surveys have shown this method to be more accurate and precise. The coefficient of variation is <7.5%.

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.


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