A Flow-Cytometric Equivalent of the Kleihauer Test

Abstract

Background and Objectives: The Kleihauer slide test is in general use to screen obstetric patients for possible fetomaternal haemorrhage. Since 1993, Rh(D)-negative patients have been tested in our laboratory by a flow-cytometric method detecting Rh(D)-positive fetal cells, a method which offers improved sensitivity and accuracy. We report another flow-cytometric method of broader application which quantitates cells according to haemoglobin F (HbF) content. Materials and Methods: The red cells are fixed with glutaraldehyde and permeabilized by exposure to Triton X-100. A polyclonal sheep antibody to HbF is incubated with the cells followed by a fluorescein-labelled anti-sheep antibody. Results: Quantitation of the percentage of fetal cells following a FMH can be achieved irrespective of the blood groups of either mother or infant, and the presence of maternal F cells need not interfere since the intensity of staining is usually less than that of fetal cells. Two of 19 transfusion-dependent patients with β-thalassaemia have been found to have red cells indistinguishable from fetal cells on the basis of HbF content, but these patients also have been found to give positive results by the Kleihauer test. Conclusions: The flow-cytometric method may serve to replace the traditional Kleihauer test since it appears to offer improved accuracy and objectivity.