Kleihauer Betke by Flow Cytometry

The Kleihauer-Betke test has been used to detect fetal to maternal hemorrhage (FMH) since Dr. Kleihauer's original publication in 1957. The classical 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. 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%.

A more accurate estimate of fetal to maternal hemorrhage is achievable using a flow cytometric method that 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 ~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%.

Specimen requirement for both methods is one lavender (EDTA) tube of maternal blood.

Reference range for the flow method is 0 - 0.09% fetal cells.

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