RI SATURN 5, Руководство пользователя

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Section 6
6
Saturn
™
Laser Systems Intended Uses
29
Research Instruments Ltd
A hole should be made around two thirds the size of the cell to be taken (see published papers for exact
dimensions) and care should be taken to drill the zona pellucida where there are no cells close by.
Again, smaller holes will give less heat exposure, so as a guideline we recommend individual hole sizes of
between 5-10µm in diameter. The laser pulse widths required to make these hole sizes will vary a little
from set-up to set-up, but will be anything in the range of between 15-1000µs (see Hole Size Selection,
page 31 ).
For blastomere biopsy, the resultant hole made in the zona should be approximately 20µm and the
opening should be made in between two blastomeres after the embryo is rotated and the cells selected
for biopsy are positioned. A hole size setting of 9-10µm should be selected and 4-6 shots are usually
sufficient to make an opening of around 20µm.
Firing the laser to make an opening in between two cells means the cells will have less exposure to the
heat generated; therefore, there will be a reduction in the chance of cell lysis and other cells in this
vicinity (above or below) being affected. The embryo should be held at the bottom of the dish when
the laser shots are being applied to maximise laser efficiency.
Trophectoderm/Blastocyst Biopsy
The aim of this biopsy is to take 5-10 trophectoderm (TE) cells from the embryo at blastocyst stage,
without causing damage to the inner cell mass (ICM). Laser assisted hatching (LAH) is performed on
day 3 or early day 5 to create a weak point in the zona pellucida through which the trophectoderm cells
can start to herniate.
If creating a hole on day 3, the hole size created should be 15µm as described for Assisted Hatching. At
day 5/6 of embryo development there should be sufficient compaction of the ICM creating herniation
of the TE. If the herniated cells are safely away from the ICM, TE cells can be aspirated with a biopsy
pipette (20-30µm ID).
If creating a hole on day 5/6, an opening of approximately 15-20µm is adequate. In this instance the
opening should be made on the side opposite to the ICM whilst the blastocyst is being held by a holding
pipette and often an immediate collapse of the blastocoelic cavity will result. The biopsy pipette should
be projected towards the collapsed trophectoderm and a few TE cells aspirated and pulled towards
the opening of the zona. To dissociate the TE cells from the remaining embryo, suction from the biopsy
pipette to the TE cells and from the holding pipette to the embryo should continue to be gently and
firmly applied to allow the laser pulses to effectively penetrate and puncture the extended TE layer.
Laser pulses of 0.4-0.8ms can be applied with a maximum of 4 shots at a time to sever the TE cells. This
series of shots can be repeated if needed.
A maximum of 5-10 cells should be aspirated when performing trophectoderm biopsy.
Blastocyst Collapse
Blastocyst collapse is a procedure to remove the blastocoelic fluid of the embryo prior to vitrification
to inhibit ice crystal formation in the cell during the freezing process. The procedure is similar to that
performed for trophectoderm biopsy at day 5/6, that is, it should be performed on expanded blastocysts
and an opening of approximately 15-20µm is adequate. The laser should be fired at the junction between
two trophectoderm cells and should be made on the side opposite to the inner cell mass (ICM). An
immediate collapse of the blastocoelic cavity will result.