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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 Rh negative women who deliver a Rh 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 useful screening method for FMH. A suspension of maternal blood is incubated with anti-D. The anti-D will bind to any Rh positive fetal RBCs present in the suspension. Indicator Rh positive cells are then added, which bind to anti-D coated fetal Rh positive cells, forming visible agglutinates (rosettes) around them. A fetal bleed of as little as 2.5 mL of fetal blood can be detected with this method. If the rosette test is positive, the degree of FMH can be quantified by using the Kleihauer Betke acid elution method or flow cytometry. The rosette test will not detect fetal RBCs with a weak D phenotype.

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, 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. Results are reported as the percent of fetal RBCs seen. The sensitivity of the method is approximately 0.1 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, thalassemias, 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 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%.

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