Detection of gamma-globin mRNA in fetal nucleated red blood cells by PNA fluorescence in situ hybridization.
OBJECTIVE
Fetal nucleated red blood cells (NRBC) that enter the peripheral blood of the mother are suitable for non-invasive prenatal diagnosis. The application of peptide nucleic acid (PNA) probes for tyramide amplified flow fluorescence in situ hybridization (FISH) detection of gamma-globin mRNA in fixed fetal NRBC is investigated.


METHODS
Hemin-induced K562 cells or nucleated blood cells (NBC) from male cord blood were mixed with NBC from non-pregnant women and analysed using both slide and flow FISH protocols. Post-chorionic villus sampling (CVS) blood samples from pregnant females carrying male fetuses were flow-sorted (2 x 10(6) NBC/sample). Y chromosome-specific PNA FISH was used to confirm that the identified gamma-globin mRNA stained cells were of fetal origin.


RESULTS
Flow FISH isolated gamma-globin mRNA positive NBCs showing characteristic cytoplasmic staining were all Y positive. The amplification system generated a population of false positive cells that were, however, easy to distinguish from the NRBCs in the microscope.


CONCLUSIONS
The gamma-globin mRNA specific PNA probes can be used for detection and isolation of fetal NRBCs from maternal blood. The method has additional potential for the study of gamma-globin mRNA levels or the frequency of adult NRBC (F cells) in patients with hemoglobinopathies.
Larsen
Rasmus Dines
RD
DakoCytomation A/S, Produktionsvej 42, DK-2600 Glostrup, Copenhagen, Denmark. rasmuslarsen@mail.dk
Schønau
Andreas
A
Thisted
Marianne
M
Petersen
Kenneth Heesche
KH
Lohse
Jesper
J
Christensen
Britta
B
Philip
John
J
Pluzek
Karl-Johan
KJ
D000328:Adult; D001483:Base Sequence; D041322:Chromosomes, Human, Y; D004900:Erythroblasts / Q000737:chemistry* / Q000166:cytology; D005260:Female; D005312:Fetal Blood / Q000737:chemistry* / Q000166:cytology; D005331:Fetomaternal Transfusion; D005434:Flow Cytometry; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics; D017404:In Situ Hybridization, Fluorescence; D007231:Infant, Newborn; D008297:Male; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D020135:Peptide Nucleic Acids / Q000032:analysis*; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D016415:Sequence Alignment; D014407:Tumor Cells, Cultured
D007074:Immunoglobulin G; D020135:Peptide Nucleic Acids; D012333:RNA, Messenger