HLA-G positive trophoblastic cells in transcervical samples and their isolation and analysis by laser microdissection and QF-PCR.
OBJECTIVE
To assess the frequency of cytotrophoblastic cells in endocervical samples collected by lavage at early stages of gestation using a specific anti-HLA-G McAb (G233). From a set of four selected samples, cells identified by immunostaining were collected by laser microdissection and then tested by quantitative fluorescent polymerase chain reaction (QF-PCR) for the presence of paternally derived DNA markers, in order to establish their fetal origin.


METHODS
Syncytial fragments and cytotrophoblastic cells from 23 transcervical samples were identified by immunostaining with McAb G233 reacting against HLA-G antigen and with antibodies against cytokeratin. Slides from the same samples were also tested by fluorescent in situ hybridization (FISH), while selected samples were analysed by QF-PCR. Slides from four samples retrieved from mothers with male fetuses were immunolabelled and then cytotrophoblastic cells, syncytial fragments and maternal epithelial cells were collected by laser microdissection and tested by QF-PCR.


RESULTS
All endocervical samples retrieved from mothers with male fetuses were found to contain some cells with chromosome Y-specific signals when tested by FISH. Using McAb anti- HLA-G, cytotrophoblastic cellular elements were detected in about 50% of the samples. From four samples, cellular elements identified by immunostaining as cytotrophoblast or syncytial fragments were collected by laser microdissection and shown to be of fetal origin when tested by QF-PCR for the presence of fetal DNA markers.


CONCLUSIONS
These results confirm that, during an early phase of gestation, fetal cells are released in the lower uterine cavity and that they can be isolated and analysed for prenatal diagnosis of single gene defects and aneuploidies.
Bulmer
Judith N
JN
Department of Pathology, Royal Victoria Infirmary, Queen Victoria Road, Newcasle-upon-Tyne, UK.
Cioni
Riccardo
R
Bussani
Cecilia
C
Cirigliano
Vincenzo
V
Sole
Francesc
F
Costa
Carlota
C
Garcia
Pilar
P
Adinolfi
Matteo
M
D000328:Adult; D002584:Cervix Uteri / Q000166:cytology*; D041322:Chromosomes, Human, Y; D004210:Dissection; D005260:Female; D006680:HLA Antigens / Q000378:metabolism*; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D006801:Humans; D007124:Immunoenzyme Techniques; D017404:In Situ Hybridization, Fluorescence; D007633:Keratins / Q000378:metabolism; D007834:Lasers; D008297:Male; D008846:Micromanipulation; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011261:Pregnancy Trimester, First; D011296:Prenatal Diagnosis / Q000379:methods*; D012732:Sex Determination Analysis; D007507:Therapeutic Irrigation; D014327:Trophoblasts / Q000166:cytology* / Q000378:metabolism
D006680:HLA Antigens; D059951:HLA-G Antigens; D015395:Histocompatibility Antigens Class I; D007633:Keratins