A flow-cytometric equivalent of the Kleihauer test

Area of Science:

• Hematology
• Immunology
• Clinical Chemistry

Background:

• The Kleihauer slide test is a standard screening method for fetomaternal hemorrhage (FMH).
• Flow cytometry has been used since 1993 to detect Rh(D)-positive fetal cells in Rh(D)-negative mothers, offering enhanced sensitivity.
• An alternative flow cytometric approach quantitating cells by hemoglobin F (HbF) content is presented.

Purpose of the Study:

• To introduce and evaluate a novel flow cytometry method for quantifying fetal cells based on HbF content.
• To assess the applicability of this method in screening for fetomaternal hemorrhage.
• To compare the accuracy and objectivity of this method with the traditional Kleihauer test.

Main Methods:

• Red blood cells are fixed with glutaraldehyde and permeabilized using Triton X-100.
• A polyclonal sheep antibody against HbF is used, followed by incubation with a fluorescein-labeled anti-sheep antibody.
• Quantification of fetal cells is based on HbF content via flow cytometry.

Main Results:

• The flow cytometry method accurately quantifies fetal cells in cases of FMH, regardless of maternal or infant blood groups.
• Maternal F cells do not interfere due to typically lower staining intensity compared to fetal cells.
• Two beta-thalassemia patients showed red cells indistinguishable from fetal cells by HbF content, but also tested positive with the Kleihauer test.

Conclusions:

• The developed flow cytometry method provides an accurate and objective alternative to the traditional Kleihauer slide test.
• This method offers improved accuracy and objectivity for screening obstetric patients for fetomaternal hemorrhage.
• The HbF-based flow cytometry technique demonstrates broader applicability in clinical settings.