Detection of gamma-globin mRNA in fetal nucleated red blood cells by PNA fluorescence in situ hybridization |
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Authors: | Larsen Rasmus Dines Schønau Andreas Thisted Marianne Petersen Kenneth Heesche Lohse Jesper Christensen Britta Philip John Pluzek Karl-Johan |
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Affiliation: | DakoCytomation A/S, Produktionsvej 42, DK-2600 Glostrup, Copenhagen, Denmark. rasmuslarsen@mail.dk |
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Abstract: | 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 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. 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. |
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