[Separation and enrichment of fetal nucleated red blood cells from maternal blood for non-invasive prenatal gene diagnosis].
OBJECTIVE
In order to develop a non-invasive technique for prenatal gene diagnosis.


METHODS
Peripheral blood mononuclear cells (MNCs) were separated by single density gradient Histopaque 1.077 from 25 pregnant women with gestations between 8 - 36 weeks. The fetal nucleated red blood cells (NRBCs) were enriched from the MNCs by positive selection using Dynabeads M-450 CD(71) or negative selection using Dynabeads M-450 CD(45). The enriched NRBCs were identified by anti-gamma-biotin or anti-zeta-biotin antibodies. Globin gene of NRBCs from fetuses with risk of beta-thalassemia major were amplified with nested PCR followed by reverse dot blot hybridization for gene diagnosis.


RESULTS
NRBCs stained by anti-gamma-biotin or anti-zeta-biotin antibodies could be found in the peripheral blood samples of the 25 pregnant women. Three out of 5 fetuses with risk of beta-thalassemia major were successfully diagnosed using these NRBCs.


CONCLUSIONS
Fetal NRBCs in maternal circulation can be isolated and enriched by single gradient density Histopaque 1.077 followed by magnetic activated cell sorting. Nested PCR can amplify DNA for gene diagnosis from no less than 20 NRBCs.
Xie
J
J
Department of Pediatrics, First Affiliated Hospital, Guangxi Medical University, Nanning 530021, China.
Long
G
G
D001710:Biotin / Q000032:analysis; D002469:Cell Separation / Q000379:methods; D004247:DNA / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004900:Erythroblasts / Q000166:cytology / Q000378:metabolism*; D005260:Female; D005312:Fetal Blood / Q000166:cytology / Q000378:metabolism; D005315:Fetal Diseases / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D005838:Genotype; D005865:Gestational Age; D005914:Globins / Q000032:analysis / Q000235:genetics*; D006801:Humans; D017493:Leukocyte Common Antigens / Q000032:analysis; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*; D017086:beta-Thalassemia / Q000097:blood / Q000175:diagnosis / Q000235:genetics
D001710:Biotin; D005914:Globins; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases; D017493:Leukocyte Common Antigens