Use of a directly conjugated monoclonal anti-D (BRAD-3) for quantification of fetomaternal hemorrhage by flow cytometry.
BACKGROUND
Determination of the volume of fetal D-positive cells in the circulation of D-negative women after delivery is carried out to determine whether additional prophylactic anti-D should be given to the mother. Although the Kleihauer-Betke test is still widely used to calculate the fetomaternal hemorrhage, increasing use is being made of flow cytometry.


METHODS
A conjugated monoclonal anti-D was prepared by labeling purified BRAD-3 (IgG3) with fluorescein isothiocyanate (FITC-BRAD-3). This reagent was used to label D-positive red cells by a one-step procedure: 5 microL of washed cells were incubated with 50 microL of FITC-BRAD-3 (50 micrograms/mL) at 37 degrees C for 30 minutes; then the cells were washed and 500,000 events were analyzed by flow cytometry.


RESULTS
The FITC-BRAD-3 reagent effectively labeled D-positive cells. The percentage of D-positive cells in mixtures containing more than 0.04 percent D-positive cells in D-negative cells was accurately determined by using this reagent and flow cytometry. Although the Kleihauer-Betke test was more accurate than this one-step flow cytometric method at quantifying fetomaternal hemorrhage of < 1 mL, the flow cytometric method was more accurate in the 1- to 7-mL fetomaternal hemorrhage range of 1 to 7 mL (whole-blood equivalents). Analysis of 175 clinical samples for fetomaternal hemorrhage gave consistent quantification results with the three methods used: the Kleihauer-Betke test, flow cytometry with FITC-BRAD-3, and flow cytometry with polyclonal anti-D followed by FITC-anti-IgG.


CONCLUSIONS
Labeling of samples with FITC-BRAD-3 was simple and rapid. By flow cytometric analysis, good separation of D-positive from D-negative cells was obtained, and fetomaternal hemorrhage of > 1 mL was quantified accurately.
Lloyd-Evans
P
P
International Blood Group Reference Laboratory, Southmead Hospital, Bristol, UK.
Kumpel
B M
BM
Bromelow
I
I
Austin
E
E
Taylor
E
E
D000911:Antibodies, Monoclonal; D005260:Female; D005331:Fetomaternal Transfusion / Q000175:diagnosis*; D005434:Flow Cytometry; D006801:Humans; D011247:Pregnancy; D012204:Rh-Hr Blood-Group System / Q000276:immunology*
D000911:Antibodies, Monoclonal; D012204:Rh-Hr Blood-Group System