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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%.

Flow cytometry can provide a much more accurate measurement of fetal red blood cells in maternal blood. Proficiency surveys have shown this method to be more accurate and precise.  The coefficient of variation is <7.5%.

This 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  more than 65,000 cells. Fetal red cells are clearly distinguished from adult cells by their significantly higher fluorescent signal. Normal adults exhibit a single peak with minimal fluorescence corresponding to HbA. Neonates have a single peak with bright fluorescence corresponding to HbF. Young infants usually have two peaks, representing both HbA and HbF.

Patients with hereditary persistence of fetal hemoglobin (HPFH) trait have a single peak of intermediate fluorescence. Other conditions with elevated levels of Hb F, such as beta thalassemia and delta-beta thalassemia have two peaks corresponding to a heterocellular pattern.

 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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