Detection of gamma-globin mRNA in fetal nucleated red blood cells by PNA fluorescencein situhybridization

Autor: Andreas Schønau, Jesper Lohse, Karl-Johan Pluzek, John Philip, Marianne Thisted, Kenneth Heesche Petersen, Rasmus Dines Larsen, Britta Christensen
Rok vydání: 2003
Předmět:
Zdroj: Prenatal Diagnosis. 23:52-59
ISSN: 0197-3851
DOI: 10.1002/pd.520
Popis: Objectives 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 × 106 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. Conclusion 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. Copyright © 2003 John Wiley & Sons, Ltd.
Databáze: OpenAIRE
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