title: Detection of gamma-globin mRNA in fetal nucleated red blood cells by PNA fluorescence in situ hybridization.
abstract: 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.
authors.affiliation: DakoCytomation A/S, Produktionsvej 42, DK-2600 Glostrup, Copenhagen, Denmark. rasmuslarsen@mail.dk
authors.forename: Rasmus Dines
authors.initials: RD
authors.lastname: Larsen
authors.forename: Andreas
authors.initials: A
authors.lastname: Schønau
authors.forename: Marianne
authors.initials: M
authors.lastname: Thisted
authors.forename: Kenneth Heesche
authors.initials: KH
authors.lastname: Petersen
authors.forename: Jesper
authors.initials: J
authors.lastname: Lohse
authors.forename: Britta
authors.initials: B
authors.lastname: Christensen
authors.forename: John
authors.initials: J
authors.lastname: Philip
authors.forename: Karl-Johan
authors.initials: KJ
authors.lastname: Pluzek
mesh_terms: 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
chemical_list: D007074:Immunoglobulin G; D020135:Peptide Nucleic Acids; D012333:RNA, Messenger