Protocol detail

PGD for Hemophilia using FISH

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From 2005 to 2009, in our center, PGD for hemophilia was based on the selection of female embryos, using FISH analysis. Embryo biopsy procedures are performed as previously reported [13 (link)–15 (link)]. Single blastomeres were fixed using Tween 20/HCl and Carnoy's solution as per the method described by Dozortsev and McGinnis [16 (link)]. Fixed slides were treated using the protocol recommended by Vysis Inc. The FISH analysis for chromosomes X, Y, 13, 18, and 21 was carried out using the Vysis MultiVysion PGT Multi-Color Probe Kit (Abbot Molecular) and following the recommendations of the manufacturers. Slides were hybridized in a HYBrite (Vysis Inc.) using the optimised FISH conditions. Posthybridization washes were performed and Antifade II counterstain (Vysis Inc.) was added. Nuclei were analysed independently by two PGD scientists using an Olympus BX51 fluorescent microscope (Olympus Optical Co. Ltd., Tokyo, Japan) and CytoVision v4.0 with the GSL-120 software.

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Fernández R.M., Peciña A., Sánchez B., Lozano-Arana M.D., García-Lozano J.C., Pérez-Garrido R., Núñez R., Borrego S, & Antiñolo G. (2015). Experience of Preimplantation Genetic Diagnosis for Hemophilia at the University Hospital Virgen Del Rocío in Spain: Technical and Clinical Overview. BioMed Research International, 2015, 406096.

Publication 2015

BX51 fluorescent microscope

Biopsy procedures Blastomeres Carnoy solution Chromosomes x Embryo Female Fish Fish conditions Hemophilia Microscope Nuclei Olympus Tween 20

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Variable analysis

independent variables

Not explicitly mentioned

dependent variables

Not explicitly mentioned

control variables

Embryo biopsy procedures were performed as previously reported [13 (link)–15 (link)].
Single blastomeres were fixed using Tween 20/HCl and Carnoy's solution as per the method described by Dozortsev and McGinnis [16 (link)].
The FISH analysis for chromosomes X, Y, 13, 18, and 21 was carried out using the Vysis MultiVysion PGT Multi-Color Probe Kit (Abbot Molecular) and following the recommendations of the manufacturers.
Slides were hybridized in a HYBrite (Vysis Inc.) using the optimised FISH conditions.
Posthybridization washes were performed and Antifade II counterstain (Vysis Inc.) was added.
Nuclei were analysed independently by two PGD scientists using an Olympus BX51 fluorescent microscope (Olympus Optical Co. Ltd., Tokyo, Japan) and CytoVision v4.0 with the GSL-120 software.

positive controls

Not specified

negative controls

Not specified

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