An investigation of methods for enriching trophoblast from maternal blood.
Trophoblast deportation is known to occur in normal human pregnancy, but it is not yet clear whether these cells routinely enter the maternal peripheral circulation and are available as a source of fetal DNA for non-invasive prenatal diagnosis of genetic disorders. To resolve this issue requires an efficient method of enriching trophoblast from maternal blood combined with a means to confirm its identity. Five different techniques were tested on ten retroplacental blood samples to determine the most sensitive and operator-efficient method. Lysis of red cells alone gave the best recovery of trophoblast but had to be discounted, together with Ficoll density gradient centrifugation, due to the very low purity and the excessive time required. Fluorescence-activated cell sorting (FACS) of pre-enriched trophoblast resulted in the lowest recovery rate (8 per cent) despite a 3250-fold enrichment and a very high purity. Immunomagnetic beads (Dynabeads) coated with anti-CD16 antibody proved to be the best method for the subsequent immunocytochemical characterization of deported trophoblast. However, IO beads coated with anti-CD45 antibody may be more useful for isolating trophoblast for prenatal diagnosis due to the high purity, enrichment (32-fold), and recovery rate (78 per cent) obtained with this method.
Johansen
M
M
Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, U.K.
Knight
M
M
Maher
E J
EJ
Smith
K
K
Sargent
I L
IL
D002452:Cell Count; D002469:Cell Separation / Q000379:methods*; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D018189:Immunomagnetic Separation; D017404:In Situ Hybridization, Fluorescence; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods; D014327:Trophoblasts / Q000166:cytology*