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PrestoCHILL
Flash Chiller for Frozen Sections
Operator Manual - MM138
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Thank you for having selected our system and welcome to the ever growing world club of users for Milestone
laboratory instrumentation.
We are sure that you will be completely satisfied with this new tool entering your laboratory.
We invite you to read carefully this operator manual and to keep it in reach for convenient and fast
consulting.
For any possible clarification or any request for assistance please contact either our Representative in your
country:
Or contact
Milestone s.r.l.
Via Fatebenefratelli, 1/5
24010 Sorisole (BG) Italy
Tel. +39.035.412 8264
Fax +39.035.575498
web site www.milestonemedsrl.com
e-mail marketing@milestonemedsrl.com
Please read the user manual before using the device.
MM138-003 – PrestoCHILL – Operator Manual
2018-05
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SUMMARY
SUMMARY ........................................................................................................................................................ 4
1. INTRODUCTION .................................................................................................................................. 6
1.1. Symbol used ...................................................................................................................................... 6
1.2. Biological and hygiene warnings ....................................................................................................... 7
1.3. Designated Uses ............................................................................................................................... 7
1.4. Technical specifications ..................................................................................................................... 7
1.4.1. Touch control terminal ............................................................................................................... 8
1.5. Transportation and storage conditions .............................................................................................. 8
1.6. Warning information ........................................................................................................................... 9
1.7. Labeling explanation ........................................................................................................................ 10
1.8. Compatible reagents ........................................................................................................................ 10
1.9. Waste disposal of the equipment .................................................................................................... 10
2. INSTALLATION .................................................................................................................................. 12
2.1. Space Requirements ....................................................................................................................... 12
2.2. Unpacking and Checking list ........................................................................................................... 13
2.3. Electrical installation ........................................................................................................................ 15
2.3.1. Power supply ........................................................................................................................... 15
2.3.2. Back side connection plate ...................................................................................................... 15
2.4. Final placement ............................................................................................................................... 16
2.5. Unit overview ................................................................................................................................... 17
2.6. Plate conformation ........................................................................................................................... 17
3. OPERATE WITH PrestoCHILL ........................................................................................................... 18
3.1. Set Date and Time ........................................................................................................................... 18
3.2. Change the language ...................................................................................................................... 18
3.3. Help .................................................................................................................................................. 19
3.4. Events log ........................................................................................................................................ 20
3.5. Service ............................................................................................................................................. 20
3.6. Start up MANUAL MODE ................................................................................................................ 20
3.7. Start up AUTOMATIC MODE .......................................................................................................... 21
3.7.1. Set automatic programs during the week ................................................................................ 21
3.8. Set up freezing parameters ............................................................................................................. 26
3.9. Defrost ............................................................................................................................................. 26
3.9.1. HEPA12 Filter .......................................................................................................................... 27
3.10. Freezing a sample for Frozen Section ............................................................................................. 28
3.10.1. Freezing time ........................................................................................................................... 31
3.10.2. Using multiple timers ............................................................................................................... 31
4. MAINTENANCE .................................................................................................................................. 33
4.1. Introduction ...................................................................................................................................... 33
4.2. Before/After each run ....................................................................................................................... 33
4.3. Daily ................................................................................................................................................. 33
4.4. Every 250 defrost pump hours ........................................................................................................ 34
4.4.1. How to change the HEPA12 filter ............................................................................................ 34
5. DECONTAMINATION AND CLEANING ............................................................................................. 36
5.1. Safety precautions ........................................................................................................................... 36
5.2. PrestoCHILL decontamination and cleaning procedure .................................................................. 36
5.2.1. Detergents ............................................................................................................................... 36
5.2.2. Procedure ................................................................................................................................ 37
6. PrestoCHILL OPTIONAL .................................................................................................................... 38
6.1. Standard mould with different measures ......................................................................................... 38
6.2. Mould for freezing in cryomould ...................................................................................................... 43
6.3. Mould for 55mm chuck .................................................................................................................... 44
6.4. Heat extractors ................................................................................................................................ 45
6.5. Box with 100 paper discs ................................................................................................................. 45
6.6. Milestone Cryoembedding Compound (MCC) ................................................................................. 45
6.7. Glass discs for MOHS procedure .................................................................................................... 46
6.8. Kit MOHS Rings (RNG-H5, REF 100880) ....................................................................................... 47
6.9. Kit Ring H25 diameter 28mm (RNG-H25-28, REF 100890) ............................................................ 48
6.10. Kit Ring H25 diameter 37mm (RNG-H25-37, REF 100891) ............................................................ 49
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A. Appendix – Tips and Tricks ................................................................................................................ 50
A.1. Fat tissues........................................................................................................................................ 50
A.2. Muscle tissue ................................................................................................................................... 52
A.3. Small Biopsies ................................................................................................................................. 53
A.4. Skin sample for MOHS with glass disc technique ........................................................................... 55
A.5. Skin sample for MOHS with ring technique ..................................................................................... 56
A.6. Freezing of high sample in vertical position ..................................................................................... 58
B. Appendix – Freezing time ................................................................................................................... 61
B.1. Recommended freezing time for moulds ......................................................................................... 61
B.2. Freezing time changes .................................................................................................................... 62
C. Appendix – Use the extractor for chucks ............................................................................................ 63
C.1. How to use the extractor for chucks ................................................................................................ 63
D. Appendix – How to change a mould ................................................................................................... 65
D.1. How to change a mould ................................................................................................................... 65
E. Appendix – Troubleshooting ............................................................................................................... 66
E.1. Block detachment from the chuck ................................................................................................... 66
E.2. Gap between sample and medium .................................................................................................. 67
E.3. Block sticking to the mould .............................................................................................................. 67
E.4. Difficulty in unscrewing moulds ....................................................................................................... 68
E.5. Power failure .................................................................................................................................... 69
F. Appendix – How to manage an alarm................................................................................................. 70
F.1. How to manage an alarm ................................................................................................................. 70
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1. INTRODUCTION
1.1. Symbol used
An instruction accompanied by this symbol provides important information and
requires more attention.
An instruction accompanied by this symbol provides a cautionary statement: failure to
follow the instruction may endanger the operator or cause damage to the instrument.
An instruction accompanied by this symbol provides a caution against electric shock.
Be sure to follow the instruction to prevent accidents resulting from electric shock.
Biohazard.
Be careful: parts identified with this symbol are zones with risks of crushing hand
and/or finger(s).
Surfaces identified with this symbol are at danger temperature.
CE logo: this instrument complies to European Community directives.
IVD medical device according to 98/79/EC directive.
Indicates a single-use medical device or one-patient use during a single procedure.
Indicates the manufacturer's batch code.
WEEE European directive symbol (2002/96/EC):
Electric/electronic device do not throw out in the environment.
Manufacturer.
Fragile.
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1.2. Biological and hygiene warnings
Please, carefully read the following note.
This instrument is an in-vitro medical device and it is installed in a location where there is presence of blood,
specimen and other biological tissues. For your safety, it is required to clean and disinfect the device before
entering in contact with it.
Also you should wear gloves when you touch it.
In addition, it is required that the unit is cleaned and disinfected before sending it to MILESTONE S.R.L.
Non-disinfected devices will no longer be accepted and you will be contacted to get the relative disinfection
cost.
In any case, international health rules forbid shipments in standard packages of biohazard materials (risk of
sanctions).
1.3. Designated Uses
The Milestone PrestoCHILL and its optional parts have been engineered for laboratory use and can
therefore withstand the hardest laboratory conditions for continuous use in freezing biological samples for
frozen sections.
The instrument is intended for laboratory use (indoor use) only.
The Milestone PrestoCHILL is specifically designed to produce frozen section blocks or frozen tissues in a
fast, reliable and standardized way.
The Milestone PrestoCHILL must be operated exclusively with the reagents listed in section “Compatible
reagents” refer to chapter 1.8.
Any other use of the instrument is considered improper and may void the warranty
provided by the manufacturer.
1.4. Technical specifications
Power supply:
PrestoCHILL REF 100851 115V~ 60Hz 250W
PrestoCHILL REF 100850 230V~ 50/60Hz 250W
Refrigerant: Helium
Time to reach -40°C (-40°F) about 40min from ambient temperature of 25°C (72°F)
Working Temperature: 15°C – 25°C (59°F – 77°F)
Humidity (relative): up to 80%
Maximum altitude: 2000m
Use temperature range: -24°C to -60°C (-11.2°F – -76°F)
Indoor use only.
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1.4.1. Touch control terminal
The following picture shows the Touch Control TERMINAL of the PrestoCHILL unit. It is possible to operate
the unit by simply touching the icons on the screen.
One USB PORT is located on the lateral sides of the Touch Control Terminal.
When not in use, it’s required to leave the USB protection inserted into the slot, as
shown in the picture above.
Characteristics: TFT-LCD 4.3” widescreen color display
Resolution 480X272,
Resistive Touchscreen
Mass storage 512MByte
1CPU 700MHz
Windows™ Compact Embedded 6
Programs: Customizable time and temperature
I/O-Interface: 1x USB port 2.0
The manufacturer reserves the right to change and/or improve specifications without notice and without
incurring any obligation.
1.5. Transportation and storage conditions
Temperature: –20°C up to +60°C (-4°F up to 140°F)
Humidity: up to 80% (up to 50% at 40°C – 104°F) non–condensing
Before turning PrestoCHILL on (when coming from a storage room), let the unit reach
working environmental conditions (at least half an hour).
It is very important to ship and move the PrestoCHILL system in a vertical position to
avoid that the cooling plate could be damaged and unscrewed from its support.
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1.6. Warning information
Should the equipment be used in different ways than those specified by the manufacturer, the warranty
provided by the manufacturer may be void.
Main power plugs are considered disconnecting device. Disconnect all plugs from the socket outlet before
assembling of the system, before connection of the optional (if present) and before cleaning.
Equipment shall be connected to a main power socket outlet which is accessible and visible by the operator.
Equipment is provided with replaceable fuses, however their replacement is to be performed by properly
authorized service personnel. If the system is not properly working, please contact the local authorized and
trained service representative of the manufacturer.
Parts of this instrument may attain temperatures as low as –42ºC. It is important to avoid allowing bare skin
to touch such cold surfaces – when in doubt, wear gloves.
All parts of the equipment and all optional must be supplied only by the manufacturer.
DO NOT OPEN THE CHASSIS. Failure to follow the instruction in the documentation
might lead to a reduction of device safety. If the equipment is not working properly,
please contact your supplier or the manufacturer.
Do not remove the instrument, optional, panels or cover. Only authorized and
qualified SERVICE personnel may repair the instrument and access the
instrument’s internal components.
It is important that normal standards of safety and good laboratory practices are
employed. Always use common sense and the best known practice when operating
the instrument.
The institution owning the instrument has primary responsibility for accident-free
operation together with designated personnel who operate, service or repair it.
To avoid damage to the instrument, use only the reagents listed in chapter 1.8
Compatible Reagents. Failure to follow the instructions in the documentation might
lead to a reduction of device safety.
Low Temperatures are present in this equipment.
Extreme care should be taken.
DO NOT let bare skin come into contact with metal surfaces.
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1.7. Labeling explanation
MILESTONE SRL
Via Fatebenefratelli, 1/5 – 24010 Sorisole (BG) –
ITALY
Tel. +39.035.4128264
Fax. +39.035.575498
PRESTO CHILL
REF
SERIAL NUMBER (SN)
FUSES
1.6AT 5X20
INPUT
MANUFACTURED:
For symbols explanation see chapter 1.1
1.8. Compatible reagents
1. Embedding medium: gel consisting of polyethylene glycol and polyvinyl alcohol which when frozen
has the same density as frozen tissue.
2. Alcohol-based detergents and polyphenolic based disinfectants to clean internal work plan and
PrestoCHILL optional.
1.9. Waste disposal of the equipment
This instrument is an in-vitro medical device and it is usually installed in a laboratory where specimens and
other biological tissues are present. For your safety, it is therefore required to clean and disinfect before
entering in contact with it. You must also wear gloves when operating the system.
In case of return it is required that the unit is cleaned and disinfected before sending it back to Milestone.
Non-disinfected devices will be no longer accepted and you will be contacted to pay the relative disinfection
cost.
International health rules require that shipments of biohazard materials are not done in standard packages
(risk of sanctions).
Do not remove the side panels of the unit during the cleaning operations.
Before doing any operation disconnect the power supply.
Do not use direct or high-pressure water to clean the instrument.
For safety reason, any operation without the individual protective devices must be
avoided.
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It is recommended to use polyphenolic based disinfectants to clean and disinfect the instrument.
For further information, please contact the manufacturer at application@milestonemedsrl.com.
Removal of equipment from use for disposal:
1) Switch OFF the unit. Disconnect the power supply.
2) Disconnect all the optional, if available.
3) Clean and disinfect all parts of the equipment according to the manufacturer specification.
4) Dispose all electric and electronic parts of the equipment.
Complying with directive 2012/19/EC of the European Parliament and of the Council of 04 July 2012 on
waste electrical and electronic equipment (WEEE), the separate collection environmental managed of
equipment is mandatory.
It is necessary to return the used equipment to the distributor or to inquire about the presence of a local
empowered system for collection and disposal of WEEE.
The in-observance of Directive 2012/19/EC or of the local law which acknowledge it can have potential
effects on the environment and human health.
This symbol indicates separate collection
for electrical and electronic equipment
If additional requirements on accident prevention and environmental protection exist in the country of
operation, this instruction manual must be supplemented by appropriate instructions to ensure compliance to
such requirements.
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2. INSTALLATION
Milestone PrestoCHILL is an instrument which requires the utmost care when it is unpacked and installed.
Provide a bench of at least 70cm (28in) in width and 70cm (28in) in depth and that can stand the
PrestoCHILL weight of 25kg (56lb).
At least 2 people should remove the unit from its box and locate it on the bench.
Then place PrestoCHILL on the bench, make sure that the unit is level and the bench is made of nonflammable material and is vibration free. Ensure that the instrument has been positioned away from hot,
direct sunlight and is in a location completely free from drafts.
Make sure the unit is not placed against any side surface limiting the air cooling inlet/exhaust: allow
at least a distance of 20cm (7.9in) for each side and of 30cm (11.8in) behind.
2.1. Space Requirements
PrestoCHILL is delivered in a wood box with these dimensions:
Width: 420mm (17in)
Height: 680mm (27in)
Depth: 650mm (26in)
Unit dimensions:
Width: 300mm (12in)
Height: 450mm (18in)
Depth: 540mm (22in)
Space dimensions required to locate the unit:
Width: 700mm (28in)
Height: 800mm (31.5in)
Depth: 700mm (28in)
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2.2. Unpacking and Checking list
When unpacking, check that all parts correspond to the packing list included in the shipment. Utmost care
must be taken during unpacking of PrestoCHILL, to avoid scratching or damage of the external coating.
The PrestoCHILL unit (230V version REF 100850, 115V version REF 100851) includes the following items:
• Qty.1 Main power cable (Code 50036 for 230V version, Code 50391 for 115V version)
• Qty.3 Mould for 30mm chuck (REF 100606-A)
• Qty.3 Mould for 40mm chuck (REF 100609-A)
• Dispensing slides:
-Qty.3 REF 100551, large tip
-Qty.3 REF 100545, small tip
• Qty.1 Magnetic pen to work with the touch-screen terminal Code 50596/S
• Qty.1 Tool for mould exchange (Code 100575)
• Tweezers:
- Qty.1 Code 100593: the small one
- Qty.1 Code 100741: the large one
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• Qty.2 Heat extractor (Code 100533)
• Qty.4 Heat extractor slim (Code 100677)
• Qty.1 Box with 100 paper discs, diameter 22mm (REF 100622)
• Qty.1 Operator manual in electronic format
• Qty.1 Chuck Extractor (Code 100737L)
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2.3. Electrical installation
2.3.1. Power supply
Power supply line must have a ground connection (yellow/green, green or bare wire).
Do not use the blue/white wire (neutral of power line). Power supply neutral has to be referred to ground.
Carefully check that:
• A Ground Connection is made available in the lab to assure the unit works correctly. Do not connect a
Milestone unit without ground connection.
• Power supply line wires (their size and distance from the switch board) used in the laboratory are
compliant with the label positioned on the back side of units.
• Plug/socket is visible during the normal use and easily accessible.
Power line protection:
• 115V: Miniature circuit breaker curve C, 6A, interruption more than 6000A.
• 230V: Miniature circuit breaker curve C, 6A, interruption more than 6000A.
Residual current device: 30mA (Class I device).
230V version: a schuko terminated power cord is provided.
115V version: a NEMA 5-15P terminated power cord is provided.
Before connecting the plug to the laboratory’s socket CHECK that the power supply
voltage of the unit matches the power supply line of the laboratory.
Damages occur when wrong power supplies the instrument.
2.3.2. Back side connection plate
On the back side of the PrestoCHILL are positioned:
- the HEPA12 filter
- the electrical plug
- the label with unit serial number
- the vent
- the spare fuses box
See also the label of the instrument that provides some important information.
Always communicate the S.N. of the unit to Customer Support.
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2.4. Final placement
PrestoCHILL unit can now be positioned in its final place of use. Make sure that the unit is level.
Then connect:
• the main power cable into the electrical plug that is located on the back of the unit (see the picture
below),
• and the plug of main power cable into the wall socket.
• Now it is possible to turn the PrestoCHILL on.
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2.5. Unit overview
1. Work area cover
2. Touch control terminal
3. Main switch
4. USB port
5. Embedding medium and spare optional
storage area
6. Cool trap
1. Heat extractors
2. Heat extractors slim
3. Moulds for 30mm chucks
4. Moulds for 40mm chucks
We advise against the use of optional other than those provided by Milestone.
2.6. Plate conformation
The work area of the PrestoCHILL houses two different types of heat extractors: standard and slim, and six
5mm deep moulds.
The standard heat extractor can be used with any type of mould among those listed in
chapter 6.
The slim heat extractor can only be used with 5mm deep moulds.
The PrestoCHILL moulds can be replaced according to the laboratory requirements: size of frozen
specimens and type of chuck used. To change the moulds see appendix D and for the list of mould models
available see chapter 6.
The conformation used by the PrestoCHILL that ensures optimal performances is: four
5mm deep moulds and two moulds with depth of your choice.
If the PrestoCHILL conformation features moulds deeper than 5mm (7 or 10mm), we recommend positioning
the slim heat extractors near the 5mm deep moulds and the standard heat extractors near the deeper
moulds.
For optimal results, Milestone recommends using 5mm deep moulds.
Do not work with a different conformation to that indicated above.
1
2
3
4
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3. OPERATE WITH PrestoCHILL
3.1. Set Date and Time
Press the “Settings” icon from the status bar. Then select “Date” and set.
3.2. Change the language
Press the “Settings” icon from the status bar. Then select “Language” and set.
Select the language and press OK.
The software needs the reboot to make the language change effective. Do it immediately, pressing “Yes” to
the below question, otherwise the language change will be operative starting from the first turn on of the unit.
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3.3. Help
In the “Help” section in the “Settings” menu, it is possible to insert useful info: serial number (only for
service), technical, application and sales assistance contacts and website.
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3.4. Events log
Clicking on “Events log” icon , allows access to the PrestoCHILL actions list.
3.5. Service
This section is allowed only for service personnel.
3.6. Start up MANUAL MODE
The time required to reach the set temperature (-40°C) from room temperature
(approximate 20°C) varies from 40 minutes to 1 hour.
Before starting, wipe the work plate of the PrestoCHILL with paper or similar absorbent
material to dry it.
Switch on the unit. This page will appear:
Touch the button “Start chiller” to set the working temperature and the sample freezing time and to initiate
the cooling power. Software records the last set temperature and time data.
The spot near the actual temperature value is red if the plate has not reached the set temperature. In this
case it is better to not freeze any samples. When the actual temperature is ±4°C the set temperature, the
spot becomes green and the plate is ready for freezing.
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3.7. Start up AUTOMATIC MODE
The time required to reach the set temperature (-40°C) from room temperature
(approximate 20°C) varies from 40 minutes to 1 hour.
Before starting, wipe the work plate of the PrestoCHILL with paper or similar absorbent
material to dry it.
Switch on the unit. This page will appear:
If you turn the unit on in a moment between Chiller start and Stop time, in 10 seconds the plate cooling starts
and the unit will be usable when the temperature reaches the set one. If you turn the unit on in another
moment, cooling will start at set time. It is always possible to modify the set temperature and the sample
freezing time, by entering in the chiller page.
For safety reasons, if the cover of the PrestoCHILL is open when the beginning of a
chiller program is set, the chiller program does not start.
3.7.1. Set automatic programs during the week
Automatic cycle is a function, which allows the user to decide the periods during the week in which the unit is
at the working temperature, and the ideal moment during each day to do the unit defrost.
Press the “Automatic cycle” icon from the Settings page. It is necessary to insert the Administrator
password (issued as a separate document inserted in the Operator Manual).
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Edit the parameters on the base of laboratory needs:
Set Chiller time:
Select the day to set up.
Select the start chiller hour, by touching on it: the hour box will have a grey border. Press on the “Start chill”
button, and select the precise starting time: at beginning, half or end of the selected hour. The selected hour
will flash.
Now select the chilling stop time, touching on it: it will become highlighted. Press the “Stop chill” button to
confirm the chiller ending time and choose the precise starting time: at beginning, half or end of the selected
hour. The period in which chiller is on will be underlined with a blue line.
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Set Defrost time:
Select the start defrost hour, by touching on it: it will have a grey border. Press on the “Start defrost” button,
and select the precise starting time: at beginning, half or end of the selected hour. The selected hour will
flash.
The software automatically calculates and reserves the needed hours for the defrost program. A standard
defrost program requires 3 hours plus 1 hour to reach the defrost temperature.
The period reserved for the defrost cycle will be underlined with a yellow line.
The chiller stop time can be selected also the day after the chiller start.
The chiller time can’t be plus than 24h, it is preferable to do a defrost cycle once a day.
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The software does not allow Chiller and Defrost programs in the same time. For example, if you try to start a
defrost program during the chiller time you will have the following message:
Or if you start a defrost program that will end during a chiller program, the message will be:
To reset all the week programs, press on the “Reset program” button, while to cancel only one program,
select it and press the “Reset selected” button, answering YES to the relative question.
Save settings, by clicking on YES to the exit question:
Activate the automatic mode by pressing the icon to render it green (active):
in the main page.
Then the main screen shows icons for use in manual mode OFF (grey color):
Defrost cycle duration depends on Milestone default settings, accessible only to Service
personnel. Contact application@milestonemedsrl.com for further information.
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During an automatic cycle, from the Home page if the current time is between the plate cooling start and end
time, the chiller screen is automatically displayed after 10 seconds:
On the left status bar, it is present the PrestoCHILL manual/automatic mode icon:
Automatic mode active
Automatic mode inactive
(Manual mode active)
With automatic mode on, if the “Settings” page or a page other than the “Home page” is
accessed, after 10 minutes of inactivity the unit returns to the home page in order to
allow the implementation of the weekly program set.
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3.8. Set up freezing parameters
IN MANUAL MODE: To set up freezing temperature and duration, enter in the Chiller page, through the
“Start chiller” icon.
IN AUTOMATIC MODE: To set up freezing temperature and duration must be in the working chiller period
(pre-set in automatic cycle settings- see chapter 3.7.1) otherwise you can not access the screen below.
When you are in the screen displayed below, modify the set temperature with the right arrows (red arrow to
increase and blue arrow to decrease) or by touching the set temperature box (in this case a keypad will
appear).
Modify the set freezing time in with the lateral arrow or by touching the Time box (in this case a keypad will
appear).
3.9. Defrost
The defrost procedure allows to obtain, in a short time, the complete elimination of frost that forms during
normal PrestoCHILL use.
Defrost procedure consists of plate heating, while an aspiration removes aqueous vapors derived from frost
evaporation.
Since samples prepared in PrestoCHILL are fresh and so potentially dangerous, defrost vapors are filtered
through an HEPA12 filter (blue arrow on the picture below), which removes biological pathogens.
Then vapors condense in the cool trap, positioned in the left angle of the PrestoCHILL frontal side, yellow
arrow on the picture below.
Milestone suggests freezing samples at -40°C.
The freezing time depends on mould size used.
Refer to chapter 3.10.1.
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When in manual mode, press the “Start defrost” icon on main screen to start defrost procedure. At the end
of this procedure the plate returns to room temperature.
In automatic mode, defrost start is already set and it will not need to be initiated by the operator.
3.9.1. HEPA12 Filter
For safety reasons, between the working area and the defrost pump, there is a HEPA12 filter that filters
possibly dangerous pathogens in the vapors.
This filter must be changed after the defrost pump has worked for 250 hours.
The software advises you that the filter life is finished with the message “Filter needs to be replaced. Please
do it”, at the starting of the first freezing program of the day.
Change the HEPA12 filter following the instructions in the chapter 4.4.1 How to change the HEPA12 filter.
Check the cool trap daily. If needed, empty it. Refer to chapter MAINTENANCE 4.3.
It is strongly suggested to do a defrost cycle once a day.
Defrost settings are under Service settings, accessible only to Service personnel.
Contact application@milestonemedsrl.com for further information.
The preset defrost temperature is 30°C, but during the cycle higher temperature values
can be reached.
Avoiding HEPA12 filter change, can be dangerous for operator safety.
To change the filter, refer to chapter 4.4.1.
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3.10. Freezing a sample for Frozen Section
Use PrestoCHILL unit to prepare blocks for frozen section.
1) Prepare the mould by cleaning with paper soaked in alcohol and placing the appropriate chuck on it
(this procedure will prevent ice inside the mould).
Cleaning of the mould is very important to avoid the block from sticking in the mould.
2) Apply a thin layer of embedding medium (MCC) to the tip of the dispensing slide. This thin layer of
MCC creates a clean plane of separation between the tissue and the mould floor and will prevent the
sticking of any tissue residue.
3) Place tissue FACE DOWN on the dispensing slide. The transparency of the dispensing slide permits
orientation of the tissue into the desired position.
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4) Look through the underside of the slide while adjusting the tissue so desired margin or other aspect
is visible in position.
5) Let sample protrude from the end of the slide and touch the leading edge of the tissue to the mould
floor. It will adhere.
6) Pull the dispensing slide out from under the tissue. Tissue requiring precise positioning can be
flattened or manipulated and guided into desired position by carrying out this step and carefully.
7) Fill the mould as quick as possible so that a meniscus of medium is bulging above the brim. Too
much medium is not a problem. Too little medium can cause failure of the specimen to anchor to the
chuck
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8) Place the chuck over the mould as quick as possible and press it.
9) Place the heat extractor over the chuck stem. The heat extractor is a nickel-plated brass block
designed to fit over the chucks. The extractor is maintained at a cold temperature to provide
additional instant and rapid heat removal from the chuck, embedding medium, and specimen.
10) Close PrestoCHILL cover.
If the cover is left open for more than 3 minutes will trigger the alarm "cover open".
Just close the cover to reset the alarm.
Alternatively press the icon that appears in the status bar to the left of the screen,
to silence the alarm.
11) Press "Start" button in "Time" screen area to begin the timer countdown.
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12) Remove the frozen specimen and chuck when timer alarms. It will be flat and the surface will require
less trimming and wastage of tissue before the entire tissue face is available for sectioning.
3.10.1. Freezing time
The freezing time depends on the size of the mould used and on the sample type.
The mould for 30 mm chuck provides a standard freezing time of 40 seconds. In case of delicate specimens
which are cut in cryostat at temperatures equal to or greater than -20°C, the freezing time can be reduced up
to 20 seconds, so as to have a less hard block and easier to cut. In case of fatty tissues we recommend
extending the freezing time to up to 80 seconds, so as to have a harder block that helps to cut the adipose
tissue.
The mould for 40 mm chuck provides a standard freezing time of 60 seconds. In case of delicate specimens
which are cut in cryostat at temperatures equal to or greater than -20°C, the freezing time can be reduced up
to 30 seconds, so as to have a less hard block and softer to cut. In case of fatty tissues we recommend
extending the freezing time to up to 120 seconds, so as to have a harder block that helps to cut the adipose
tissue.
3.10.2. Using multiple timers
The “multiple timers” button on the Chiller page opens a screen with 6 timers:
More than one timer can be useful in some cases:
-When it is necessary to freeze more samples simultaneously.
-When different size moulds are present in the PrestoCHILL.
-When wanting to freeze different tissues in the PrestoCHILL with specific times.
For freezing times of differently shaped or sized moulds, see appendix B.
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The 6 timers are modified and used like the individual timer (see chapter 3.8).
The name of the timer can be modified: click on the name to display the alphanumeric keyboard and enter
the desired name (maximum 13 digits).
The multiple timers page cannot be accessed if the timer of the main page is running.
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4. MAINTENANCE
4.1. Introduction
Maintenance of PrestoCHILL is carried out at two levels:
The first is at laboratory level: a simple routine maintenance can prevent the likelihood of more serious
problems. Minimal routine maintenance is required by laboratory personnel that use PrestoCHILL.
The second level of maintenance is technically more detailed but can be carried out from normal laboratory
personnel.
4.2. Before/After each run
Ensure that PrestoCHILL is generally kept clean after each use. Do not use abrasives or sharp instruments
that may damage the coating.
Before each run cleaning of the mould is very important to avoid the block from sticking
in the mould, cleaning must be performed with paper soaked in alcohol.
4.3. Daily
It’s required to check the level of water condensed in the condensation trap, daily.
As you can see below, the maximum level of liquid is labeled on condensation trap. When the level is near
the limit, remove the cooltrap pulling it and empty any collected fluids. Then reinsert the condensation traps.
Make sure that the condensed liquid does not go over the limit indicated.
A decontamination procedure must be performed daily, please refer to chapter 5 DECONTAMINATION AND
CLEANING.
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4.4. Every 250 defrost pump hours
For safety reasons, between the working area and the defrost pump, there is a HEPA12 filter that filters
possibly dangerous pathogens in the vapors.
This filter must be changed after the defrost pump has worked for 250 hours.
The software advises you that the filter life is finished with the message “Filter needs to be replaced. Please
do it”, at the starting of the first freezing program of the day.
4.4.1. How to change the HEPA12 filter
For safety reasons, after the defrost pump has worked for 250 hours, the HEPA12 filter (Code 100624) must
be changed. The filter is on the rear of the unit.
1. Extract the metal connection of the filter hose from the unit cover.
2. Take out the second filter hose from its fitting.
Avoiding HEPA12 filter change, can be dangerous for operator safety.
Dispose of the HEPA filter according to current regulations.
For safety reasons, any operation without the required personal protection equipment is
to be avoided.
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3. Remove the filter from its housing, leveraging with the finger.
4. Insert the new filter into the housing, being sure that the filter direction (see the blue arrow on the
filter) corresponds with the black arrow on the unit.
5. Now insert the metal part of the long hose into the cover hole then connect the other filter hose to its
place.
After changing the filter, reset the defrost pump hour counter.
Click on the icon from the Settings page. It is necessary to insert the Administrator password
(issued as a separate document inserted in the Operator Manual). Then press YES to the below question:
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5. DECONTAMINATION AND CLEANING
The procedure described in this chapter must be performed daily.
For safety reasons, any operation without the required personal protection equipment is
to be avoided.
PrestoCHILL decontamination is mandatory due to the biological hazard, in view of the
use of fresh specimens.
5.1. Safety precautions
The personnel involved in the decontamination procedure of PrestoCHILL and its optional (if present) must
wear the appropriate individual protective devices, in particular:
• Single use, HI-RISK protective gloves (100% hi-risk nitrile or equivalent). Individual protection device
that meets the standard EN 374 and the Directive 89/686/CEE.
• Antibacterial mask face for surgery room and white coat. Individual protection device, meets the
standard EN 149 Grade FFP3D and the Directive 89/686/CEE.
5.2. PrestoCHILL decontamination and cleaning procedure
5.2.1. Detergents
The following instructions must be strictly complied with in order to perform proper decontamination and
cleaning procedure.
For further information, contact our specialists at the following email address:
application@milestonemedsrl.com.
Do NOT remove the unit's side panels during cleaning operations.
For decontamination and cleaning do not use procedures other than the one explained in
this chapter.
Before any operation, disconnect from the power supply mains. Do not use direct or high
pressure water to clean the instrument.
It is recommended to use disinfectants containing polyphenols.
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Germicidal efficacy of polyphenolic based disinfectant agents:
5 minutes: Immunodeficiency Virus Type 1 (HIV-1), Hepatitis B Virus (HBV), Hepatitis C Virus
(HCV), Rotavirus, Coronavirus SARS.
15 minutes: Staphylococcus Aureus, Candida Albicans.
30 minutes: Pseudomonas Aeruginosa, Mycobacterium Bovis (TB).
5.2.2. Procedure
1) Cold Plate, moulds, heat extractors, dispensing slide and tweezers
• Disinfect plate, mould and heat extractor with polyphenol-based reagent, leaving it to work for the time
necessary as explained above and, if necessary, remove any organic traces using a non-abrasive tool.
• Rinse with a cloth moistened with water
• Clean with ethanol (concentration in the range 70 – 100%)
• Dry with adsorbent paper or a clean cloth.
2) Chucks
• Rinse chucks under tap water until all freezing medium (OCT) traces go away.
• Wash with an appropriate alcohol-based detergent.
• Dry with adsorbent paper or a clean cloth.
3) Final steps
• Place the positioning tool, chucks and heat extractors on the cold plate.
• Before removing gloves, wash them with soap, water and disinfectant.
• Remove the individual protection devices.
• Wash your hands.
• Make sure the contaminated material is packed and disposed in an approved incinerator in compliance
with all federal, provincial and local government regulation.
On completion of the cleaning operations, wait at least 5 minutes before connecting the
power supply cable.
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6. PrestoCHILL OPTIONAL
Not included, to be ordered apart:
We advise against the use of optional other than those provided by Milestone.
6.1. Standard mould with different measures
MLD3228 2926-5
rectangular shape
chuck of 26 x 31mm
depth 5mm, weight 0.062kg
max sample size 26,5 x 23,5mm
REF 100590
MLD3228 2926-7
rectangular shape
chuck of 26 x 31mm
depth 7mm, weight 0.059kg
max sample size 26 x 23mm
REF 100694
MLD3030 2525-5
square shape
chuck of 28 x 28mm
depth 5mm, weight 0.063kg
max sample size 22.5 x 22.5mm
REF 100693
MLD3030 2525-7
square shape
chuck of 28 x 28mm
depth 7mm , weight 0.060kg
max sample size 22 x 22mm
REF 100698
MLD3030 2525-10
square shape
chuck of 28 x 28mm
depth 10mm , weight 0.056kg
max sample size 21 x 21mm
REF 100699
MLD3939 3333-5
square shape
chuck of 37 x 37mm
depth 5mm, weight 0.054kg
max sample size 31,5 x 31,5mm
REF 100692
MLD3939 3333-7
square shape
chuck of 37 x 37mm
depth 7mm, weight 0.048kg
max sample size 31 x 31mm
REF 100696
MLD3939 3333-10
square shape
chuck of 37 x 37mm
depth 10mm, weight 0.040kg
max sample size 30 x 30mm
REF 100697
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MLD4129 3324-5
rectangular shape
chuck of 40 x 28mm
depth 5mm, weight 0.058kg
max sample size 30,5 x 21,5mm
REF 100666
MLD4129 3324-7
rectangular shape
chuck of 40 x 28mm
depth 7mm, weight 0.055kg
max sample size 30 x 21mm
REF 100700
MLD4129 3324-10
rectangular shape
chuck of 40 x 28mm
depth 10mm, weight 0.050kg
max sample size 29 x 20mm
REF 100701
MLD4234-5
round shape
chuck of 35mm
depth 5mm, weight 0.054kg
max sample size 31,5mm
REF 100702
MLD4234-7
round shape
chuck of 35mm
depth 7mm, weight 0.050kg
max sample size 31mm
REF 100703
MLD4234-10
round shape
chuck of 35mm
depth 10mm, weight 0.044kg
max sample size 30mm
REF 100704
MLD3022-5
round shape
chuck of 25mm
depth 5mm, weight 0.064kg
max sample size 19,5mm
REF 100605-A
MLD3022-7
round shape
chuck of 25mm
depth 7mm, weight 0.062kg
max sample size 19mm
REF 100705
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MLD3426-5
round shape
chuck of 30mm
depth 5mm, weight 0.061kg
max sample size 23,5mm
REF 100606-A
MLD3426-7
round shape
chuck of 30mm
depth 7mm, weight 0.059kg
max sample size 23mm
REF 100607-A
MLD3426-10
round shape
chuck of 30mm
depth 10mm, weight 0.055kg
max sample size 22mm
REF 100608-A
MLD4436-5
round shape
chuck of 40mm
depth 5mm, weight 0.052
max sample size 33,5mm
REF 100609-A
MLD4436-7
round shape
chuck of 40mm
depth 7mm, weight 0.047kg
max sample size 33mm
REF 100610-A
MLD4436-10
circular shape
chuck of 40mm
depth 10mm, weight 0.040kg
max sample size 32mm
REF 100611-A
MLD3330 2419-5
rectangular shape
chuck of 26 x 31mm
depth 5mm, weight 0.065kg
max sample size 21,5 x 16,5mm
REF 100713
MLD3330 2419-7
rectangular shape
chuck of 26 x 31mm
depth 7mm, weight 0.063kg
max sample size 21 x 16mm
REF 100714
MLD3330 2419-10
rectangular shape
chuck of 26 x 31mm
depth 10mm, weight 0.060kg
max sample size 20 x 15mm
REF 100715
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MLD4442 4436-5
oval shape
chuck of 40mm
depth 5mm, weight 0.050kg
max sample size 33,5 x 39,5mm
REF 100716
MLD4442 4436-7
oval shape
chuck of 40mm
depth 7mm, weight 0.044kg
max sample size 33 x 39mm
REF 100717
MLD4442 4436-10
oval shape
chuck of 40mm
depth 10mm, weight 0.036kg
max sample size 32 x 38mm
REF 100718
MLD4410-5
round shape
chuck of diameter or diagonal 20 - 40mm
depth 5mm, weight 0.064kg
max sample size 7,5mm
REF 100707
MLD4410-7
round shape
chuck of diameter or diagonal 20 - 40mm
depth 7mm, weight 0.064kg
max sample size 7mm
REF 100708
MLD4410-10
round shape
chuck of diameter or diagonal 20 - 40mm
depth 10mm, weight 0.064kg
max sample size 6mm
REF 100709
MLD4415-5
round shape
chuck of diameter or diagonal 20 - 40mm
depth 5mm, weight 0.063kg
max sample size 12,5mm
REF 100710
MLD4415-7
round shape
chuck of diameter or diagonal 20 - 40mm
depth 7mm, weight 0.063kg
max sample size 12mm
REF 100711
MLD4415-10
round shape
chuck of diameter or diagonal 20 - 40mm
depth 10mm, weight 0.062kg
max sample size 11mm
REF 100712
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MLD2618-5
round shape
chuck of 22mm
depth 5mm, weight 0.066kg
max sample size 15,5mm
REF 100727
MLD2618-7
round shape
chuck of 22mm
depth 7mm, weight 0.065kg
max sample size 15mm
REF 100728
MLD4326 1818-5
square shape
chuck of 25 x 25mm
depth 5mm, weight 0.067kg
max sample size 15.5 x 15.5mm
REF 100730
MLD4326 1818-7
square shape
chuck of 25 x 25mm
depth 5mm, weight 0.065kg
max sample size 15 x 15mm
REF 100731
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6.2. Mould for freezing in cryomould
MLD50 2520-5
rectangular shape
cryomould of 25 x 20mm
compatible with chuck of diameter or diagonal 20 - 55mm
depth 5mm, weight 0.070kg
REF 100726
MLD5017-5
round shape
cryomould of 17mm
compatible with chuck of diameter or diagonal 20 - 55mm
depth 5mm, weight 0.071kg
REF 100767
MLD5025-5
round shape
cryomould of 25mm
compatible with chuck of diameter or diagonal 20 - 55mm
depth 5mm, weight 0.074kg
REF 100768
MLD50 1616-5
square shape
cryomould of 16 x 16mm
compatible with chuck of diameter or diagonal 20 - 55mm
depth 5mm, weight 0.074kg
REF 100769
Use of moulds for freezing in cryomould
1. Prepare the specimen in cryomould at room temperature and fill the latter with MCC.
2. Transfer to mould MLD50 2520-5 and add chuck and standard heat extractor. Freeze for a time
between 30 and 150 seconds, depending on the cryomould dimensions and then check that the
block is completely frozen. If needed wait more time.
3. Remove the cryomould from the block and cut in cryostat or detach the block from the chuck and
store it in the freezer.
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Due to plastic insulation, freezing with cryomould is slower and could lower the end
quality.
If the PrestoCHILL is used only for cryomould freezing, it is possible to lower the
temperature in order to have a faster freezing. For further info, please, contact
application@milestonemedsrl.com.
6.3. Mould for 55mm chuck
MLD5951-5
round shape
chuck of 55mm
depth 5mm, weight 0.083kg
max sample size 48,5mm
REF 100722
MLD5951-7
round shape
chuck of 55mm
depth 7mm, weight 0.073kg
max sample size 48mm
REF 100723
You can use a single mould MLD5951 in the working area of the PrestoCHILL.
If using a MLD5951 mould the PrestoCHILL conformation allows the presence of 3
moulds: one MLD5951 mould in position 4 and two standard sized moulds in position 1
and 5.
With this type of moulds it is not advisable to use the slim heat extractor. Always use the
standard heat extractor, see next paragraph.
Avoid several consecutive freezing cycles with this type of mould.
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6.4. Heat extractors
HEAT EXTRACTOR STANDARD
circular base with diameter hole of 12mm
Code 100533
HEAT EXTRACTOR SLIM
circular base with diameter hole of 12mm
Code 100677
HEAT EXTRACTOR RECTANGULAR
rectangular base 20 x 30mm
Code 100592
HEAT EXTRACTOR LARGE OPENING
circular base with diameter hole of 17mm
Code 100736
6.5. Box with 100 paper discs
- 16mm diameter (REF 100623)
- 22mm diameter (REF 100622)
- 30mm diameter (REF 100615)
6.6. Milestone Cryoembedding Compound (MCC)
Milestone Cryoembedding Compound (MCC) 125ml (REF 51421 for the 12 bottle package)
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6.7. Glass discs for MOHS procedure
- Hand protection tool for MOHS glass discs of 22 and 32mm diameter (Code 100790)
- Qty 10 MOHS glass disc with 22mm diameter and 1.5mm thickness (REF 100870)
To be used with moulds: MLD3426-5, MLD3426-7 and MLD3426-10
- Qty 10 MOHS glass disc with 32mm diameter and 1.5mm thickness (REF 100871)
To be used with moulds: MLD4436-5, MLD4436-7 and MLD4436-10
- Qty 10 MOHS glass disc with 48mm diameter and 1.5mm thickness (REF 100872)
To be used with moulds: MLD5951-5 and MLD5951-7
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6.8. Kit MOHS Rings (RNG-H5, REF 100880)
- Flat metal plate RNG-PLATE (REF 100881)
- Conductive pad for metal plate (Code 100882)
- Qty 3 – RNG7028-5
Ring with diameter of 28mm and thickness of 5mm (REF 100883)
For chuck of at least 30mm
- Qty 3 – RNG7037-5
Ring with diameter of 37mm and thickness of 5mm (REF 100884)
For chuck of at least 40mm
- Ring extractor (Code 100885)
- Ring extractor bases
For ring of internal diameter of 28mm (Code 100886)
For ring of internal diameter of 37mm (Code 100887)
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6.9. Kit Ring H25 diameter 28mm (RNG-H25-28, REF 100890)
- Flat metal plate RNG-PLATE (REF 100881)
- Conductive pad for metal plate (Code 100882)
- RNG7028-25
- Ring with diameter of 28mm and thickness of 25mm (REF 100888)
- For chuck of at least 30mm
- Ring extractor (Code 100885)
- Ring extractor base for ring of internal diameter of 28mm (Code 100886)
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6.10. Kit Ring H25 diameter 37mm (RNG-H25-37, REF 100891)
- Flat metal plate RNG-PLATE (REF 100881)
- Conductive pad for metal plate (Code 100882)
- RNG7037-25
- Ring with diameter of 37mm and thickness of 25mm (REF 100889)
- For chuck of at least 40mm
- Ring extractor (Code 100885)
- Ring extractor base for ring of internal diameter of 37mm (Code 100887)
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A. Appendix – Tips and Tricks
A.1. Fat tissues
Fatty tissues are difficult to section using a cryostat because they are non-aqueous materials and simply do
not freeze. The problem is clear when the fat begins to smear and impedes cutting of any tissue in its path.
To make fat hard enough to section, it must be cooled to a very low temperature that often is too cold for
non-fatty components present in the same sample. Another problem is that fat does not adhere well to the
embedding medium.
Milestone studied a new protocol that allows fat cutting.
1) Prepare the mould by cleaning with paper soaked in alcohol and placing the appropriate chuck on it
(this procedure will prevent ice inside the mould).
Cleaning of the mould is very important to avoid the block from sticking in the mould.
2) Prepare the sample on the spatula with thin layer of MCC medium under it. If preferable, it is
possible to use the paper disk method (see BOP 102).
3) Transfer the sample inside the mould and immediately fill the mould with MCC medium.
4) Put chuck and heat extractor.
5) Freeze for 4 minutes at -40°C.
6) Trim the block in the cryostat and try to get a thin slice. It is possible that during the trimming the
block temperature became too warm to section.
7) Put a mark on the block in order to recognize the position of the block inside the cryostat.
8) Cool down the block inside the PrestoCHILL for 1 minute. Be sure to put the block surface in contact
with the PrestoCHILL cold plate: not in the mould used for the freezing because the thickness of the
block now is reduced, it is better to use a larger mould or a free space on the metal plate.
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9) After the 1 minute re-cooling, attach the chuck in its cryostat housing in the same position used for
the trimming.
10) Cut immediately in order to get a slice before the warming up of the block. It is very important to cut
the block in quickly, moving the cryostat wheel very fast.
11) If, during cut, the section curls up, flatten it using two small brushes.
12) Pick up the section and fix immediately.
It is possible to repeat the cooling down of the trimmed block as needed to obtain a good frozen slice.
Milestone suggests the use of MCC medium for better adhesion between the sample and embedding
medium. Thanks to its higher fluidity, MCC flows faster to surround and adhere to the samples.
If some non-fatty parts are present in the sample and they shatter, it is possible to warm these parts up with
gloved thumb.
Last problem about fat is that this material tends to adhere less to the glass and then to came off during
staining. In this case, it is possible to try warming the glass microscope slide before picking up the slice or
cut a thinner slice.
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A.2. Muscle tissue
Muscle tissues are very rich in water and this causes the formation, during freezing, of large ice crystals
within and between the muscle fibres. In addition, it is important in order to morphologically evaluate the
muscle tissues to have a cross section.
The PrestoCHILL is used to freeze the muscle specimen quickly and in the correct position, through a
technique different from the standard one. Instead to put the specimen on the spatula and move it inside the
mould through a sliding movement, the sample must be put directly into the mould.
1. Prepare a drop of MCC on the tip of the spatula. Take the specimen with tweezers so as to have the
section of interest free and wet it with the drop of MCC.
2. Place the specimen in the mould so that the muscle fibres are vertical, this will provide cross section
orientation for the cut.
3. Holding the specimen firmly, fill the mould with MCC. Then place the chuck and heat extractor and
wait for the freezing time to elapse.
4. Extract the block and cut and stain the slide.
At the end of the freezing and cutting processes, it is possible to follow the BOP 105
procedure in order to further improve the quality of the muscle tissue sections.
Mould with smaller well give a faster freezing and consequently a better quality.
See code MLD4415 and MLD4410.
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A.3. Small Biopsies
The difficult part in small biopsies freezing is to put them in the same level. In fact, they tend to collapse
inside the medium and consequently it is necessary to do many slices to view the entire biopsy or all the
biopsies. In this case, we suggest the paper embedding:
1) Prepare the mould by cleaning with paper soaked in alcohol and placing the appropriate chuck on it
(this procedure will prevent ice inside the mould).
2) Apply a thin layer of embedding medium (MCC) to the tip of the dispensing slide. Place a circle of
lens paper on the MCC drop and press it in order to wet the paper, then flip the paper over and place
it on the drop of medium, wetting both sides. It is better to leave an edge of the paper without OCT in
order to have a not sticky handle for the tweezers during sliding.
We suggest the use of blue lens paper because the blue colour gives the right contrast to better
understand if the sample is correctly positioned and oriented.
3) Place tissue FACE DOWN and in the needed position and orientation. More than one piece of tissue
can be put on one paper square according to the size, as shown in the photo above.
4) Touch the leading edge (preferably without MCC) of the paper to the mould floor, block the paper to
the mould floor with the forceps. It will adhere to the cold mould. Then pull the dispensing slide out
from under the paper.
Avoid taking the paper disk with tweezers to drag it into the mould because this makes the
positioning of the disc in the center of the base mould more difficult.
During this phase it is very important to use tweezers with cleaned and thin tips, otherwise they may
stick to the paper, making difficult its correct positioning into the mould.
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5) Fill the mould so that a meniscus of medium is bulging above the brim. Too much medium is not a
problem. Too little medium can cause failure of the specimen to anchor to the chuck.
6) Place the chuck over the mould as quickly as possible and press it. Place the heat extractor over the
chuck stem. Now close PrestoCHILL cover and press on countdown Start button and wait pre-set
freezing time.
7) Take the block out. It will be flat with the paper over the specimen. The entire sample, in case of long
thin biopsy, or all samples, in case of multiple short biopsies, will be at the same level and specimen
will be visible through the blue lens paper due to its semi-transparency. Moreover trimming will be
easier because when the paper goes away, it means that the sample is ready to cut.
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A.4. Skin sample for MOHS with glass disc technique
The difficult part in skin sample freezing is to put it with all its margins in the same level and so in one slide.
Milestone studied a method that, through the use of glass, allows to fix all the margins to the flat glass, in
order to have them at the same level in the frozen block.
1) Relax and ink the sample, following the standard lab procedure.
2) Put the glass disc inside the dedicated spot of the hand protection tool (available only for 22 and
32mm discs) or hold the 48mm disc directly with the fingers.
3) Put the sample on the glass disc with the epidermis up and in the needed position. Push down and
adhere sample margins to the glass.
4) If necessary, press down one site of the margin with the finger and freeze it, cryospraying the
corresponding position from the other side of the glass. Repeat for all the margins.
5) Put the glass disc with the sample inside the PrestoCHILL mould and fill the mould with MCC.
6) Put chuck and heat extractor and freeze at -40°C for the suggested time for the used mould (see
appendix B).
7) Detach the glass disc from the block, heating it with the palm for few seconds.
8) Trim and cut in the cryostat.
Each glass disc can be used only for one sample.
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A.5. Skin sample for MOHS with ring technique
The difficult part in skin sample freezing is to put it with all its margins in the same level and so in one slide.
Milestone studied a method that, through the use of a planar and cold surface, allows to fix all the margins to
the flat plate, and then thanks to the ring, allows the inclusion of the leveled margins sample.
1) Remove from the PrestoCHILL working area the heat extractors and the six moulds, unscrewing
them. Insert the pad and then the ring plate with the serigraphy down. Add the needed tools.
2) Relax and ink the sample following lab procedures.
3) Clean plate and ring, using ethanol moistened paper, just before to use them.
4) Put the sample on the cleaned plate. Press all the lateral margins with tweezers or fingers, in order
to adhere them to the plate. The absence of the mould allow an easier manipulation of the sample.
Before to freeze the sample on the plate, check that the position of the sample allows to put
the chosen ring around it.
The space needed for the ring is:
- 20mm for the RNG7037-5
- 25mm for the RNG7028-5.
Don’t put samples on the external border of the plate.
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5) Put the ring around the sample.
6) Fill the internal part of the ring with MCC. Start from sample margins in order to fix them immediately.
7) Put the chuck centering the ring hole and then heat extractor and wait for the freezing time, Use the
screen timer to check to count the freezing time. 60 seconds for the 37mm ring and 40 seconds for
the 28mm ring. In the meantime, close the cover.
8) Open the unit and remove the heat extractor with the frozen block attached.
9) To detach the block from the ring it’s possible to use the ring extractor.
10) Choose the right base depending on the ring dimension. Put the ring on top of the extractor base.
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11) Put the extractor over the ring and push down in order to move up the block and outside the ring.
If block extraction is difficult, warm the base of the block
with a gloved hand. This will soften residual MCC that is
formed under the ring.
12) Trim and cut in the cryostat.
A.6. Freezing of high sample in vertical position
This procedure is for the freezing of samples long up to 2.5cm that must be frozen in vertical position. For
example, in case of rat or mouse brain that must be cut in frontal (coronal) sections, see picture below:
Milestone suggests to freeze sample inside high ring at a temperature of -80°C. This allows a
fast enough freezing also of big samples.
1) Remove from the PrestoCHILL working area the heat extractors and the six moulds, unscrewing
them. Insert the pad and then the ring plate with the serigraphy down. Add the needed tools.
Because this type of ring is high, to use the heat extractor and close the PrestoCHILL cover
during the freezing, it is necessary to remove the handle from the heat extractor.
It works correctly enhancing the freezing of the sample.
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1) Clean plate and ring, using ethanol moistened paper, just before to use them.
1) Place the high ring on the top of the cold plate and insert the sample inside it. Maintain the sample in
the needed position with the tweezers.
2) Fill the ring hole with MCC (Milestone Cryoembedding compound) up to 2mm under the edge.
3) Place the heat extractor over the ring and close the PrestoCHILL cover. Wait the freezing time and
then check the freezing completion lifting the heat extractor.
The freezing time depends on the dimension and the sample type, it is :
- 120 seconds for RNG7028-25 at -80°C
- 180 seconds for RNG7037-25 at -80°C
4) At the end of the freezing time, add MCC to completely fil the ring hole and place on it the chuck and
the heat extractor. Freeze for 20 seconds in order to attach the block to the chuck.
5) Open the unit and remove the heat extractor with the frozen block attached.
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6) To detach the block from the ring it’s possible to use the ring extractor. Choose the right base
depending on the ring dimension. Put the ring on top of the extractor base.
7) Put the extractor over the ring and push down in order to move up the block and outside the ring.
Complete the extraction pushing the block.
If block extraction is difficult, warm the base of the block
with a gloved hand. This will soften residual MCC that is
formed under the ring.
8) If the freezing was made at -80°C, leave the block in the cryostat for 15minutes to ambient it. Trim
and cut the block.
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B. Appendix – Freezing time
B.1. Recommended freezing time for moulds
Freezing times (seconds)
Heat extractor standard
Heat extractor Slim
Non-intensive
use
Intensive
use
Intensive
use
MLD3022-5
10
30
40
MLD3022-7
30
40
MLD3426-5
30
45
60
MLD3426-7
60
70
MLD3426-10
80
90
MLD4436-5
45
50
60
MLD4436-7
75
75
MLD4436-10
180
180
MLD3330 2419-5
30
45
50
MLD3330 2419-7
60
70
MLD3330 2419-10
80
90
MLD4234-5
45
50
60
MLD4234-7
75
75
MLD4234-10
180
180
MLD3228 2926-5
40
50
60
MLD3228 2926-7
70
75
MLD3030 2525-5
40
50
60
MLD3030 2525-7
70
75
MLD3030 2525-10
130
135
MLD3939 3333-5
45
50
60
MLD3939 3333-7
75
75
MLD3939 3333-10
180
180
MLD4129 3324-5
40
50
60
MLD4129 3324-7
70
75
MLD4129 3324-10
130
135
MLD4442 4436-5
45
50
60
MLD4442 4436-7
75
75
MLD4442 4436-10
180
180
MLD4410-5
10
10
10
MLD4410-7
15
15
MLD4410-10
15
15
MLD4415-5
15
15
15
MLD4415-7
20
20
MLD4415-10
25
25
MLD50 2520-5
30
45
50
MLD50 1616-5
25
40
45
MLD5025-5
25
40
45
MLD5017-5
20
35
40
MLD2618-5
10
30
40
MLD2618-7
30
40
MLD4326 1818-5
10
30
40
MLD4326 1818-7
30
40
MLD5951-5
90
MLD5951-7
150
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Intensive use of the PrestoCHILL means a use that involves the possibility of executing at least 5
consecutive freezing cycles.
Non-intensive use means a use in which each freezing cycle is spaced out by at least 15 minutes of
inactivity.
The freezing times in the table are indicative and can vary according to the conditions of
the laboratory and of the embedding medium used.
Milestone recommends using Milestone Cryoembedding Compound (MCC).
The freezing times must be confirmed by each laboratory. For further information, please
contact application@milestonemedsrl.com.
B.2. Freezing time changes
The ideal freezing time depends on the size of the mould used but also on the type of frozen tissue.
In case of delicate specimens which are cut in cryostat at temperatures equal to or greater than -20°C, the
freezing time can be reduced to halve the time suggested in the above table, so as to have a less hard block
and better suited to the cut.
In case of fatty tissues we recommend extending the freezing time to double the freezing time suggested in
the above table, so as to have a harder block that helps to cut the adipose tissue.
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C. Appendix – Use the extractor for chucks
At the end of freezing the block could stick to the mould, as a result of one of the following factors:
• Frost inside the mould before its use. In this case ice forms between the block and the mould that
acts as glue between the two parts.
Cleaning the mould is very important to prevent the block from sticking to the mould.
• Use of warm embedding medium (temperature above 25°C). In this case ice may form in the
embedding medium layer in contact with the mould.
• Using moulds deeper than 5mm: when preparing specimens in 7 and 10mm deep moulds it is more
difficult to extract the block because the contact surface between block and mould is larger.
• Using embedding medium other than Milestone Cryoembedding Compound.
Milestone recommends using embedding medium Milestone Cryoembedding Compound
(MCC - Code 51420).
For optimal results, Milestone recommends using 5mm deep moulds.
C.1. How to use the extractor for chucks
• Loosen, by rotating anti-clockwise, the jaw stop, so that the jaws widen more than the diameter of
the chuck handle
• Insert the jaws under the chuck handle
• Close and secure the jaws by rotating the stop clockwise
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• Pull the extractor for chucks towards the other, so as to perform a vertical movement. Do not tilt the
extractor for chucks.
• Loosen, by rotating anti-clockwise, the jaw stop to free the chuck with the block.
The chuck extractor is used to extract the chuck if this has a handle: in case of flat
chucks, you can use the connection to the cryostat, in case of chucks with pin, there is
no handle unless the o-ring is removed in order to use the o-ring slot as connection for
the chuck extractor.
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D. Appendix – How to change a mould
D.1. How to change a mould
On the basis of the blocks size needed in the laboratory, it is possible to change the mould on the cold plate.
• Using the key for the changing of the mould, unscrew the wrong size mould.
• Screw the desired mould into place and tighten with the key.
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E. Appendix – Troubleshooting
E.1. Block detachment from the chuck
At the end of freezing, it may happen that the MCC block with the specimen, when detaching from the chuck,
remains inside the mould.
Solution
In this case, add MCC on the block and re-fit the chuck and heat extractor. Wait 30 seconds then remove the
block by pulling the chuck vertically.
How to avoid
1. Clean the mould with paper soaked in ethanol before its use.
2. Make sure to place a suitable amount of embedding medium to reach the clamping engravings of the
chuck: better to put excess embedding medium rather than not enough.
3. Extend the freezing time.
4. Make sure that the embedding medium residues are removed from the used chuck and that the
engravings for clamping to the MCC are not worn. If the chuck does not have optimum clamping, it is
best to keep it at room temperature (outside the working area of the PrestoCHILL) before its use.
This allows the MCC to have more time, before freezing, to penetrate the clamping engravings.
5. Prefer the standard heat extractor that induces faster freezing in the clamping area between
embedding medium and chuck.
6. Prefer embedding medium Milestone Cryoembedding Compound (MCC) which, being more fluid,
penetrates better the clamping engravings of the chuck.
7. Do not use the embedding medium at a temperature above 25°C.
8. Avoid extracting the chuck by tilting it, pull with a vertical movement. Use the extractor for chucks
provided with the unit. For its use, refer to appendix C.
9. Prefer the use of 5mm deep moulds to deeper ones.
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E.2. Gap between sample and medium
The filling of the mould with medium must be done immediately after the sample transfer. If not, the sample
starts to freezing and doesn’t link with the medium. The gap between sample and medium make the cut
difficult.
Solution
Trim the block until the gap is gone away.
How to avoid
1. Fill the mould with medium immediately after the sample is transferred inside the mould.
2. Make sure to have an available bottle with enough medium to fill the mould at the right moment.
3. Prefer Milestone Cryoembedding Compound (MCC) which, being more fluid, adheres better to the
sample.
E.3. Block sticking to the mould
At the end of freezing, it may happen that the block doesn’t exit from the mould. This happen when there is
frost inside the mould that act as a glue between mould and MCC block.
Solution
Vertically hit the chuck with the heat extractor. This will break the link between block and mould and make
the block free. Don’t use the tweezers like lever.
If the removing it is very difficult, unscrew the mould and put it under cold water for very few seconds, in
order to melt the linking layer and detach the frozen block.
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How to avoid
1. Clean the mould with paper soaked in ethanol just before its use.
2. If you need ready moulds, clean them with paper soaked in ethanol, and cover them putting the
relative chucks. The chuck will avoid the formation of frost inside the mould, until its use.
3. Do not use the embedding medium at a temperature above 25°C.
4. Prefer the use of 5mm deep moulds to deeper ones.
E.4. Difficulty in unscrewing moulds
If some medium goes under the mould, it acts as a glue between the mould and the PrestoCHILL working
area and makes difficult to unscrew the mould for cleaning procedures.
Solution
Start a defrost program in order to heat the medium under the mould.
Vertically hit the mould with heat extractor, in order to break the links between mould and plate.
Unscrew the mould.
How to avoid
1. Avoid to put too much medium inside the mould, otherwise it goes under the mould.
2. Clean the PrestoCHILL working area before to start a defrost program.
3. Remove the medium pieces, if present, from it.
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E.5. Power failure
In the event of a power failure during a chiller program, when the power returns, PrestoCHILL turns on again
and goes back to the home page.
If in manual mode (see chapter 3.6) the chiller program must be restarted by the user.
If in automatic mode (see chapter 3.7) the chiller program restarts after 10 seconds.
In the event of a power failure during a defrosting process in manual mode, when the power returns, the
defrosting process does not restart and the following screen appears:
It is not possible to start with a chiller program: the software informs that it is necessary to run a defrosting
program to clean the working area of the PrestoCHILL.
Press OK and start a defrosting program. Alternatively, dry and thoroughly clean the working area of the
PrestoCHILL and all optional before starting a chiller program.
Before starting, wipe the work plate of the PrestoCHILL with paper or similar absorbent
material to dry it.
In the event of a power failure during a defrosting process in automatic mode, when the power returns, the
defrosting process does not restart and any chiller programs are blocked. The following screen will appear:
Press OK and start a manual defrosting program. Alternatively, dry and thoroughly clean the working area of
the PrestoCHILL and all optional and wait for the PrestoCHILL to run the preset automatic cycle (paragraph
3.7.1).
Before starting, wipe the work plate of the PrestoCHILL with paper or similar absorbent
material to dry it.
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F. Appendix – How to manage an alarm
F.1. How to manage an alarm
CODE
DESCRIPTION
WHAT TO DO
1
Communication fault
1. Press ‘Silent’ to hide the alarm.
2. Reboot the system. (wait 2 minutes before the rebooting)
3. Call your customer service if the issue can’t be fixed.
2
Chiller temperature
not reached
3
Defrost temperature
not reached
6
Chiller system error
7
Chiller temperature
out of range
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marketing@milestonemedsrl.com
www.milestonemed.com
Via Fatebenefratelli, 1/5 - 24010 Sorisole (BG) - Italy
Tel +39 035 4128264 - Fax +39 035 575498
Milestone Srl
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