Asymptote EF600 Series User manual

EF600 Series Instruction Manual
Asymptote nitrogen-free controlled rate freezer
Asymptote Ltd
St John's Innovation Centre
Cowley Road
Cambridge CB4 0WS
Tel: [phone] Fax: [phone]
e-mail: [e-mail]

EF600 Instruction Manual
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Tel: [phone], Fax: [phone], E-mail: [e-mail]
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Contents
1. INTRODUCTION ................................................................................................5
1.1 WARNING..............................................................................................................6
1.2 SAFETY PRECAUTIONS...........................................................................................6
1.3 MAINTENANCE.......................................................................................................6
2. INSTALLING THE FREEZER UNIT ...............................................................7
2.1 UNPACKING ...........................................................................................................7
2.2 POSITIONING THE FREEZER....................................................................................7
2.3 ELECTRICAL CONNECTION ....................................................................................7
2.4 SWITCHING THE FREEZER ON .................................................................................7
4. USER INTERFACE (EF600-01, EF600-10).....................................................10
4.1 SELECTING THE LOGGING INTERVAL (EF600-01, EF600-10) AND FREEZING
PROFILE (EF600-10) ............................................................................................10
4.2 LOG FILE NAMES (EF600-01, EF600-10).............................................................11
5. OPERATING PROCEDURE FOR THE EF600 FREEZER .........................13
5.1 PRE-OPERATION CHECKS......................................................................................13
5.2 PREPARING THE NUCLEATING TOOL .....................................................................13
5.3 PREPARING THE STRAWS......................................................................................13
5.4 LOADING THE STRAWS ........................................................................................13
5.5 STARTING THE DATA LOGGING (EF600-01, EF600-10) .......................................14
5.6 STARTING THE FREEZING CYCLE ..........................................................................14
5.7 SAMPLE NUCLEATION..........................................................................................15
5.8 END OF CYCLE .....................................................................................................15
5.9 STOPPING THE DATA LOGGING .............................................................................16
5.10 RESTARTING THE CYCLE ......................................................................................16
5.11 CLEANING THE UNIT ............................................................................................16
6. FREEZING CYCLES ........................................................................................17
6.1 HUMAN EMBRYO CRYOPRESERVATION ................................................................17
6.2 HUMAN OOCYTE CRYOPRESERVATION .................................................................17
6.3 HUMAN BLASTOCYST CRYOPRESERVATION .........................................................18
6.4 HUMAN EMBRYONIC STEM CELL CRYOPRESERVATION.........................................18
6.5 HUMAN SPERMATOZOA CRYOPRESERVATION –LINEAR COOLING ........................19
6.6 HUMAN SPERMATOZOA CRYOPRESERVATION –NON-LINEAR COOLING ................20
7. REFERENCES....................................................................................................21

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APPENDIX 1: TROUBLESHOOTING ..................................................................21
A1.1 FREEZING CYCLE WILL NOT START ....................................................................22
A1.2 NO POWER TO UNIT ...........................................................................................22
A1.3 DATA LOGGING DOES NOT FUNCTION ................................................................22
A1.4 STRAWS STUCK TO SAMPLE PLATE ....................................................................22
APPENDIX 2: INSTALLING ASYMPTOTE’S DATA LOGGING SOFTWARE
......................................................................................................................................24
A2.1 SYSTEM REQUIREMENTS ...................................................................................24
A2.2 INSTALLING THE PROGRAM ...............................................................................24
APPENDIX 3: UNINTERRUPTIBLE POWER SUPPLY (UPS).........................29
APPENDIX 4: CRYOPEN NUCLEATING TOOL OPERATION......................29

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List of Figures
Figure 3 – Desktop shortcut icon...................................................................................9
Figure 4 – User interface..............................................................................................10
Figure 4.1 – Freezing profile .......................................................................................11
Figure 4.2 – Log file names .........................................................................................12
Figure 5.4 – Straws positioned in sample plate ...........................................................14
Figure 5.7 – Nucleation of straws................................................................................15
Figure 6.1 – Pre-programmed human embryo freezing cycle .....................................17
Figure 6.2 – Pre-programmed human oocyte freezing cycle.......................................18
Figure 6.3 – Pre-programmed human blastocyst freezing cycle..................................18
Figure 6.4 – Pre-programmed human embryonic stem cell freezing cycle .................19
Figure 6.5 – Pre-programmed human sperm freezing cycle........................................19
Figure 6.6 – Pre-programmed human spermatozoa non-linear freezing cycle............20
Figure A2.1 – Installation box 1 ..................................................................................24
Figure A2.2 – Installation box 2 ..................................................................................25
Figure A2.3 – Installation box 3 ..................................................................................25
Figure A2.4 – Installation box 4 ..................................................................................26
Figure A2.5 – Installation box 5 ..................................................................................26
Figure A2.6 – Installation box 6 ..................................................................................27
Figure A2.7 – Installation box 7 ..................................................................................27
Figure A2.8 – Installation box 8 ..................................................................................28

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1. Introduction
There are three versions of the Asymptote controlled rate freezer:
EF600-00: Stand alone freezer with no data logging capacity.
The EF600-00 is pre-loaded with a single default program for the
cryopreservation of human embryos (Lasalle et al., 1985). An alternative
program may be specified at the time of ordering.
EF600-01: Freezer with data logging to a dedicated PC (supplied by
Asymptote Ltd.).
The EF600-01 is pre-loaded with a single default program for the
cryopreservation of human embryos (Lasalle et al., 1985). An alternative
program may be specified at the time of ordering.
EF600-10: Freezer with data logging to a dedicated PC (supplied by
Asymptote Ltd.).
The EF600-10 is pre-loaded with six standard freezing programs (embryo,
oocyte, blastocyst, stem cell, spermatozoa linear program and spermatozoa
non-linear program), which the user selects in the supplied data logging
software. Additional programs, up to a total of 10, may be specified at the
time of ordering.
This manual includes operating instructions for all three versions. The following
optional extras may also be supplied with the EF600 series freezer:
Cryopen nucleating tool (EF-611)
Uninterruptible power supply (UPS) (EF-613)
Conversion kit to allow portability (EF-614)
Data logging software for upgrading the EF600-00 (EF-620) and EF600-01
(EF-621)
Software for programming linear temperature profiles (EF-622)
Software for programming non-linear temperature profiles (EF-023)
Operating instructions for these optional extras are given in Appendices 2 - 7.

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1.1 Warning
It is essential that this instruction manual is read before using the EF600
freezer. Particular attention should be paid to the safety precautions
below.
1.2 Safety precautions
Operating the freezer
Temperatures down to -100ºC are reached within this equipment. Ensure that
protective gloves are worn when accessing the freezing plate, during and after
the freezing cycle, to avoid cold burns. Do not move the freezer whilst
operating as the baseplate will be hot.
Operating the cryopen nucleating tool
Read the instruction manual and safety instructions supplied with the cryopen
nucleating tool. Ensure that gloves are worn during use and never point the
cryopen towards the face.
Plate temperature
The plate temperature should never exceed 30ºC. If it does, turn the freezer
off immediately.
1.3 Maintenance
A service contract on the EF600 series freezers is available. Contact
[email protected] for full details. Please note that there are no user
serviceable parts within the EF600 and that breaking the security tags
invalidates any warranties.

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2. Installing the Freezer Unit
2.1 Unpacking
Carefully unpack the Asymptote EF600 controlled rate freezer from its carton
and place upon a suitable bench. Only lift the freezer by placing hands
underneath the freezer; do not move the freezer by pressing on the casework.
Inspect the unit to ensure that no damage has occurred during transit. The
following items should be included in the carton:
EF600-00:
a) EF600 freezer unit and lid
b) Instruction manual – on CD
c) Power lead
EF600-01 and EF600-10:
a) EF600 freezer unit
b) Instruction manual – on CD
c) Power lead
d) Communications cable
e) Laptop computer with data logging software for either EF600-01 or
EF600-10 installed
f) CD back up of installed software
Optional extras, such as a cryopen nucleating tool and UPS, will have been
supplied with the EF600 freezer if ordered.
If the unit shows any sign of damage, or if any of the items listed above are
missing, please contact the supplier immediately.
2.2 Positioning the freezer
Ensure the freezer is positioned so that the vents at the side of the freezer unit
and the air outlet at the rear of the freezer unit are not blocked. Leave a
clearance of at least 200 mm.
2.3 Electrical connection
For normal connection to the mains, plug the power lead into the rear of the
freezer unit and connect to an appropriate single-phase mains supply. If an
UPS is being used please refer to Appendix 3. If operating as a portable unit
please read Appendix 5 before using the freezer.
2.4 Switching the freezer on

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Connect the communications lead (9-pin D-type male to female) between com
port 1 (the serial port) on the computer and the back of the freezer. Power-up
the freezer unit using the ON/OFF switch on the front panel of the unit (figure
2.4). The switch illuminates red when the power is switched on.
Figure 2.4 – Front panel of freezer unit (keypad and ON/OFF switch)
3. Starting Asymptote’s Data Logging Software (EF600-01,
EF600-10)

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Start the software by either double-clicking on the shortcut icon on your
desktop (figure 3) or by selecting ‘Asymptote Data Logger’ from the program
list in the start menu on your computer.
Figure 3 – Desktop shortcut icon
The program window will open. Initial values for the ‘Set Point
Temperature’, ‘Plate Temperature’ and ‘Stirling Voltage (V)’ will be
displayed as ‘999.00’; these will automatically update once communications
between the freezer and the computer have been established.

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4. User Interface (EF600-01, EF600-10)
The current ‘Set Point Temperature’ of the freezer is displayed in red on the
left of the screen. This is the required temperature of the sample plate.
The current ‘Plate Temperature’ is displayed in blue in the centre of the
screen. This is the actual temperature of the sample plate.
The current ‘Stirling Voltage (V)’ is displayed in green on the right of the
screen. This is the output level of the Stirling Engine and is only provided for
diagnostic engineering purposes only.
The graph can show either the last minute of the cycle or the whole run.
Check ‘last minute’ or ‘whole run’ to switch between these features. The time
elapsed and the total run time of the cycle is displayed in the top left hand
corner of the user interface. The bottom of the user interface displays
information on the status of the cycle.
Figure 4 – User interface
4.1 Selecting the logging interval (EF600-01, EF600-10) and freezing profile

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(EF600-10)
When communications have been established (user interface is no longer
reading 999) select the logging interval and the desired freezing program from
the drop down menus (figure 4.1). 10 seconds is suggested as a default for the
logging interval.
Figure 4.1 – Freezing profile
4.2 Log file names (EF600-01, EF600-10)
Choose profile
(EF600-10)
Choose
logging
interval
(EF600-01,
EF600-10)

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Data is stored in the file indicated on the user interface (figure 4.2). The
default file name is ‘asydatafile’. This file name can only be changed when
the program is not logging by clicking on the grey ‘SPECIFY FILE PATH’
button.
Choose a clear and distinguishable file name for the logged data. Do not
include ‘\’, ‘/’, ‘*’ or any other punctuation marks in the file name. Do not
include a file name extension. The default extension is ‘.csv’. Files are also
automatically logged as encrypted files with the extension ‘.aef’.
If a file already exists with the file name chosen, any logged data will be
added to the end of the existing file; previous data will not be overwritten.
Figure 4.2 – Log file names
Log File
Name

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5. Operating Procedure for the EF600 Freezer
5.1 Pre-operation checks
Before operating, ensure that:
a) The freezer has been installed and switched on (section 2).
b) The data logging software has been opened (EF600-01, EF600-10 section 3.2)
and communications established (no longer reading 999).
c) The appropriate file name for the logged data has been selected (EF600-01,
EF600-10 section 4.1).
d) The desired logging interval (EF600-01, EF600-10) and freezing profile has
been selected (EF600-10 section 4.1).
5.2 Preparing the nucleating tool
The cryopen is a sterile medical device that can be used to nucleate the sample
straws. If the cryopen has been ordered with the freezer, please refer to
appendix 4 for instructions on how to prepare and use it.
Alternative tools for nucleating (e.g. forceps) may be used if sterility of the
operating environment is not essential. These should be placed in liquid
nitrogen before the freezing cycle is started.
5.3 Preparing the straws
Prepare up to 12 0.25ml standard IMV straws as normal and seal with a straw
plug, crytaseal, PVA powder or other approved sealant. Thoroughly dry the
outside of the straws after preparation to prevent them from freezing to the
sample plate.
5.4 Loading the straws
After the freezer has been switched on it takes approximately 10 minutes for
the temperature to stabilize. When the freezer has reached a stable
temperature the ‘Ready To Load’ green light will be illuminated.
Remove the lid completely and check that the sample plate is dry. If there is
any condensation on the plate, gently dry it with tissues or a soft cloth. Never
dry the plate with a hot-air gun, as this could damage the freezer, or with a
material that could scratch the plate surface.
Position the straws into the sample plate and fully press them into the grooves
(figure 5.4). Place the lid over the sample plate checking that it is located

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correctly.
Figure 5.4 – Straws positioned in sample plate
5.5 Starting the data logging (EF600-01, EF600-10)
To start the data logging, click on the grey ‘START LOGGING’ button on the
user interface. A message box will appear, prompting the user to check that an
appropriate file name has been given. To go back and check, press ‘Cancel’;
to start logging, click on ‘OK’.
5.6 Starting the freezing cycle
When the straws have been positioned correctly, wait for at least one minute
before starting the freezing cycle to allow the straws to reach thermal
equilibrium with the plate. Note that the freezer must also have been switched
on for at least two minutes in order for the controller to ‘initialise’.
Start the freezing cycle by pressing the ‘Start Cycle’ button on the keypad.
The ‘Cycle On’ button will illuminate green and will remain on for the entire
freezing cycle.
Note that the ‘Ready To Load’ light may remain on for a short time at the

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same time as the ‘Cycle On’ light. It will switch off automatically when the
temperature starts to decreases.
5.7 Sample nucleation
When the samples reach a programmed hold at -7 ºC (the nucleation plateau) a
buzzer sounds and the ‘Nucleate Now’ yellow light illuminates. Nucleation
must be done at this time. The program allows a 10 minute period for the
samples to be nucleated, unless otherwise specified.
Remove the lid of the freezer completely. Touch the nucleating tool (if using
the cryopen please refer to Appendix 4 for operating instructions) to the first
straw for approximately 1 second (figure 5.7). Repeat with the remaining
straws and close the lid of the freezer. Turn off the buzzer by pressing the
‘Buzzer Off’ button.
Inspect the straws approximately one minute after nucleation to verify that
there is some ice-growth in the straws. If ice has not formed in any of the
straws repeat nucleation.
Figure 5.7 – Nucleation of straws
5.8 End of cycle
When the cycle is complete the yellow ‘Cycle Complete’ light will illuminate.
The straws can now be removed from the freezer and placed in storage. The

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freezer will hold at the final temperature of the cycle until it is switched off.
5.9 Stopping the data logging
When all of the straws have been transferred, click on the grey ‘STOP
LOGGING’ button on the user interface. A message box will appear,
confirming that the user wants to stop logging. Click on ‘OK’ to stop logging
(or ‘Cancel’ to continue logging).
The log file is automatically stored as a .csv (comma separated variables) file,
in the folder specified. The default location for the ‘Logged Files’ folder is:
‘C:\Program Files\ Asymptote\ Asymptote Data Logger\’.
To view the log files, locate the appropriate file in Windows Explorer, right
click on it and choose ‘Open With’, selecting ‘Excel’ from the available
options. Alternatively the file may be opened directly from Excel.
The encrypted file (.aef extension) should be saved separately in an audit
folder.
5.10 Restarting the cycle
The freezer must be switched off and switched back on again before another
freezing cycle can run. Switch the freezer off using the ON/OFF switch at the
front of the freezer, leave the lid on and wait for the plate to warm up to room
temperature. Dry the sample plate between runs. Turn the freezer back on and
allow the temperature to stabilise.
For a faster turn around between runs switch the freezer off and then back on
again. Monitor the plate temperature and, when it reaches 10ºC, turn the
freezer back off again for a few minutes. Dry the plate, turn the freezer on
again and wait for the ‘Ready To Load’ light to turn on.
5.11 Cleaning the unit
The freezer unit and sample plate is designed to be easily cleaned and
sterilised. The surface of unit and the sample plate can be wiped down with a
sterile filtered Biocide C wipe. Dry the sample plate thoroughly after
cleaning.

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6. Freezing cycles
The freezing cycles correspond to the standard set of freezing programs pre-
loaded in the EF600 freezer. Unless otherwise requested, equipment pre-
loaded with a single program (EF600-00 and EF600-01) will run the standard
profile for human embryo cryopreservation (section 6.1). The EF600-10
freezer is loaded with all six profiles below (sections 6.1 to 6.6).
6.1 Human embryo cryopreservation
• Start temperature 20°C
• Cool from 20°C to -7°C at 2°C min-1
• Hold for nucleation at -7°C for 10 minutes
• Cool from -7°C to -30°C at 0.3°C min-1
• Cool from -30°C to -100°C at 10°C min-1
• Hold at -100°C
-100
-80
-60
-40
-20
0
20
00:00 00:20 00:40 01:00 01:20 01:40 02:00
Time (hh:mm)
Temperature (°C)
Figure 6.1 – Pre-programmed human embryo freezing cycle
6.2 Human oocyte cryopreservation

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• Start temperature 20°C
• Cool from 20°C to -8°C at 2°C min-1
• Hold for nucleation at -8°C for 10 minutes
• Cool from -8°C to -30°C at 0.3°C min-1
• Cool from -30°C to -100°C at 10°C min-1
• Hold at -100°C
-100
-80
-60
-40
-20
0
20
00:00 00:20 00:40 01:00 01:20 01:40 02:00
Time (hh:mm)
Temperature (°C)
Figure 6.2 – Pre-programmed human oocyte freezing cycle
6.3 Human blastocyst cryopreservation
• Start temperature 20°C
• Cool from 20°C to -7°C at 1°C min-1
• Hold for nucleation at -7°C for 10 minutes
• Cool from -7°C to -30°C at 0.3°C min-1
• Hold at -30°C
-30
-20
-10
0
10
20
00:00 00:20 00:40 01:00 01:20 01:40 02:00
Time (hh:mm)
Temperature (°C)
Figure 6.3 – Pre-programmed human blastocyst freezing cycle
6.4 Human embryonic stem cell cryopreservation

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• Start temperature 20°C
• Cool from 20°C to -7°C at 2°C min-1
• Hold for nucleation at -7°C for 10 minutes
• Cool from -7°C to -80°C at 1°C min-1
• Hold at -80°C
-80
-60
-40
-20
0
20
00:00 00:20 00:40 01:00 01:20 01:40 02:00
Time (hh:mm)
Temperature (°C)
Figure 6.4 – Pre-programmed human embryonic stem cell freezing cycle
6.5 Human spermatozoa cryopreservation – linear cooling
• Start temperature 20°C
• Cool from 20°C to -7°C at 2°C min-1
• Hold for nucleation at -7°C for 10 minutes
• Cool from -7°C to -100°C at 10°C min-1
• Hold at -100°C
-100
-80
-60
-40
-20
0
20
00:00 00:10 00:20 00:30 00:40 00:50
Time (hh:mm)
Temperature (°C)
Figure 6.5 – Pre-programmed human sperm freezing cycle

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6.6 Human spermatozoa cryopreservation – non-linear cooling
• Start temperature 20°C
• Cool from 20°C to -7°C at 2°C min-1
• Hold for nucleation at -7°C for 10 minutes
• Cool non-linear from -7°C to -100°C at an
approximate rate of 10°C min-1
• Hold at -100°C
-100
-80
-60
-40
-20
0
20
00:00 00:10 00:20 00:30 00:40
Time (hh:mm:ss)
Temperature (°C)
Figure 6.6 – Pre-programmed human spermatozoa non-linear freezing cycle