GUIDELINES FOR BLASTOMERES
COLLECTION AND TRANSPORTATION
REAGENTS AND EQUIPMENT
? Attenuated glass pasteur pipettes
? Sterile PCR Tubes (0.2 ml)*
? Alkaline Lysis Buffer* (store at -20?C; single use
? Sterile Rack*
? Mineral Oil*
? Sterile Gloves
*These materials will be provided by our Centre.
EMBRYO BIOPSY AND TRANSFER OF BLASTOMERES IN PCR TUBES
1. Embryo biopsy should be performed in sterile conditions
in IVF lab.
2. The fundamental criteria for the selection of the
blastomeres should be the presence of a clear nucleus and
the maintenance of cell integrity.
3. The biopsy of 2 blastomeres from each embryo is
advisable. If the embryo ≤6 cells stage biopsy can be
performed for one cell only.
4. Biopsied blastomeres should be manteined in drops of Ca++
Mg++ free biopsy medium, before transferring them into PCR
5. The biologist performing the transfer of the blastomeres
into the PCR tubes must use bonnet, mask and sterile gloves.
6. Attenuated glass Pasteur pipettes with 60-100μm diameter
can be used for blastomeres transfer. These pipettes should
be prepared before biopsy with a flame source and stored
under UV until their use. Commercial pipettes are also
available for this purpose. Minimum one pipette for each
embryo is necessary.
7. After removal, each blastomere should be washed twice in
drops of Ca++ Mg++ free medium, then should be carefully
aspirated by using the glass pipette under stereo microscope
with 2-3 μl of medium. Afterwards, blastomeres should gently
transferred into sterile 0.2 ml PCR tubes, containing 5 ?l
of alkaline lysis buffer (provided by our Centre), under
stereo or phase contrast inverted microscope and overlaid
with one drop of PCR mineral oil (provided by our Centre).
IMPORTANT: Blastomeres should be transferred into PCR tubes
with as less as possible amount of medium (no more than
2-3μl). An exceeding quantity of medium could determine PCR
8. All steps should be double-checked, with a second
biologist witnessing the correct labelling of dishes and
9. The pipettes must be changed after the transfer of
blastomeres of each embryo (one pipette for each embryo
should be used).
10. Each biopsied blastomere should be transferred into
separate PCR tubes. Each tube should be labelled with the
number of the embryo and a letter indicating the blastomere
(for example, 1A for blastomere A of embryo 1; 1B for
blastomere B of embryo 1). Use an indelible pen to label the
11. Tubes containing 1-2 ?l of medium, collected from wash
drop of each blastomere and from cell-free wash drop, should
added to PCR tubes containing 5 ?l of alkaline lysis buffer
(provided by our Centre), to be used as negative (blank)
controls (one blank per blastomere, and one final medium
blank for the whole set). Add to the tubes one drop of PCR
mineral oil (provided by our Centre).
12. Spin down the tubes using a microcentrifuge for few
seconds and place them in the sterile rack.
13. Seal the rack with parafilm and place it in a safety box
or envelope for transportation.
14. Remember to fill the PGD acceptance form
Suggestions for contamination control
Stringent precaution should be taken in order to minimize
contamination occurrences. Gloves and dedicated lab coats
should be worn during preparation of single cells
Preparation of media and reagents should be carried out
within a laminar flow cabinet located in a ?clean? room
restricted to those activity. Mineral oil pipetting should
be performed using aerosol-resistant pipette tips and a
dedicated pipette for this purpose.
Gloves should be changed very frequently. Hood surface, and
pipetters should be cleaned with chlorine bleach or absolute
ethanol and treated with UV light before uses.
? One specimen tube open at a time.
? Avoid carry-over of lyses buffer to next cell. If cell
lyses, change pipet tips and biopsy a second cell in a
separate tube labeled accordingly.
? Do not use oil.
? No stage warmer.
? Limit the number of embryos biopsied at one time to 5 to
prevent evaporation of placement droplet and wash droplet.
? Limit the transfer of media into wash droplet.
? Keep the sample tubes in an upright orientation holds the
DNA in a set position through shipping for accurate DNA
? Check tube labels are readable before packing tubes.
? Include original informed consent forms (yours and ours).
? Include embryo biopsy worksheet.
? Keep a supply of cold shipping packets and Styrofoam
containers that come with reagents.
? Avoid dry ice.
? The day before biopsy, request courier pickup an hour
after expected completion time.
? Fax biopsy data worksheet ahead of shipment.
? Attach airline security letter on your letterhead to
outside of box.