Quantitating fetomaternal hemorrhages of D+ red cells using an FITC-conjugated IgG monoclonal anti-D by flow cytometry: a case report

Abstract

Several methods for quantitating fetomaternal hemorrhages (FMHs) have been described; these include the Kleihauer-Betke and red cell rosetting tests, and flow cytometry that uses an indirect antiglobulin technique, employing either FITC-conjugated IgG/unlabeled anti-D or streptavidin conjugates with biotinylated anti-D to enumerate D+ red cells in maternal blood. We have used a recently described directly conjugated FITC anti-D for direct flow cytometric (direct FC) quantitation of FMH in a patient who presented with a large fetal bleed (approx. 80mL) as determined using the Kleihauer method. We compared the efficacy of the direct FC technique to the roset- ting and Kleihauer tests in estimating the quantity of Rh immunoglobulin (RhIg) to be administered to the mother to suppress Rh alloimmunization. Both the Kleihauer and the direct FC gave precise estimates of 80mL for the size of bleed, whereas the rosetting test failed to be as precise. The former tests predicted that a 10,000 iu dose (2,000 μ g) of RhIg would be adequate; the lack of alloanti-D in a maternal follow-up sample obtained 9 months after delivery supported this prediction and underlined the reliability of the direct FC method as an alternative to the Kleihauer method for quantitating large FMHs.