cellsprint user guide model cs24 and cs48 v4 - bionova | tec and c… · user guide genial genetic...

User Guide

Genial Genetic Solutions Ltd P.O. Box 3751, Chester, CH1 9UF, U.K.T +44 (0) 1244 404970 F +44 (0) 1244 404971 www.genialgenetics.com

Document Properties

Author(s) Genial Genetic Solutions

Revision 4.5

Date 10 Feb 2013

Model CS24 and CS48

Serial Number

http://www.genialgenetics.com

http://www.genialgenetics.com

Copyright NoticeThis document is the copyright of Genial Genetic Solutions Ltd. It contains proprietary information that is disclosed solely for the informing and reference purposes of clients of Genial Genetic Solutions Ltd. The contents of this document shall not, in whole or in part: (i) be used for any other purposes (ii) be disclosed to any member of the client’s organisation not having a need to know such information, nor to any third party individual or organisation or (iii) be stored on any retrieval system or be reproduced or transmitted in any form by photocopying or any optical, electrical, mechanical or other means without proper written permission of the directors of Genial Genetic Solutions Ltd.

CellSprint User Guide

© Genial Genetic Solutions 2013 i

Table of Contents

Introduction 1

Features 2

A - Safety 2

B - Sample Security 3

C - Operation 4

Aspiration Notes 5

Liquid Sensing 5

Collision Sensing 5

Fix Mix Bottle (OPTIONAL FEATURE) 6

Difference between Resuspension and Delivery 7

Delivery Volumes 7

Delivery and Resuspension Dispensing Rates 7

Parts Nomenclature 8

A - Exterior 8

A4 - Castors 8

A6 - Heating & Air Filtration 8

A7 - Control Panel 9

Main Processing Chamber 10

B1 - Carousel (in 48 place models only) 10

B2 Turntable/Centrifuge Cover (in 48 place models only) 11

B3 Centrifuge 11


© Genial Genetic Solutions 2013 ii

B5.1 Gripper Tower Assembly (in 48 place models only) 12

B5.2, B5.3, B5.4: Dispensing Towers 13

B4.1, B4.2 Wash Stations 14

Reagents Chamber 15

Reagents Chamber Vessels 16

Aspirant Waste Vessel 16

Schematic of fluid linkages 17

Initial Checks A 18

Initial Checks B 20

Setup 21

Protocols: Description, Management 22

Delivery, Resuspension, Pellet Depth 22

Parameters 22

Relationship between incubation and spin times 22

Managing Protocols 24

Loading Samples 26

Balancing the centrifuge 27

Regular Operations A 28

Making Checks 28

Declare Number of Buckets to be processed 28

Choose a protocol 29

Datuming Operation 29

Monitor Progress, or walk away 29

Bucket Geometry 30

Main Cycle Messages 30


© Genial Genetic Solutions 2013 iii

Sub Cycle Status Messages 30

Main Commentary Report 31

Regular operations B: Bench Document 32

Manual Control 33

Notes on manual control: 35

Periodic Maintenance 36

Daily 36

Weekly 36

Monthly 36

Yearly 36

Emptying the Vacuum Bottle 37

Appendix A: HMI Error Messages 38

Reacting to Alarms in General 38

Error Messages 38

Appendix B: Technical Specification 40

Power 40

Humidity & Temperature 40

Weight 40

Size 40

Air Filtration 40

Reagent Bottles 40

Test Tubes 40

Appendix C: EC Declaration of Conformity 41


© Genial Genetic Solutions 2013 iv

IntroductionThe MultiPrep CellSprint is designed to automate centrifugation, aspiration, re-suspension and addition of reagents to effect fully optimised laboratory suspension culture harvesting according to any of 16 protocols, all of which are fully user-configurable.

It will process between 1-24 samples per batch in a start-and-walk-away mode. Samples (in standard 12ml to 15ml centrifuge tubes) are inserted into centrifuge buckets which are loaded on the centrifuge rotor. These samples are indexed by the MultiPrep CellSprint so that the progress of each sample can be tracked at every stage. The centrifuge buckets will accommodate a range of conical or hemispherical - ended sample tubes, as well as admitting Leighton culture tubes in specific orientations.

Hypotonic and fixative solutions are injected automatically according to the protocol being used. If auto-fix-mix option is fitted, fresh fixative solution is automatically made for each iteration of the protocol and the proportion of methanol to acetic acid within the fixative solution can be varied from phase to phase. The volume of reagents required is determined by the protocol used and the number of samples being processed. If weigh-scales option is fitted, the MultiPrep CellSprint monitors the levels of reagents and waste and issues warnings when thresholds are reached. Other warnings are given, if for example the centrifuge becomes unbalanced, or if the system detects or encounters any unexpected events whilst performing routine tasks.

A range of useful standard protocols is pre-configured for the MultiPrep CellSprint. However, these can be amended as required and new protocols can be configured by the client using a touch-screen pad. Once set, these protocols are called and implemented automatically. In addition, any protocol can be “stepped through” manually to explore the effects of a particular setting, for example, in an experimental protocol, or to finish a harvest if the automatic program was aborted for some reason. The instrument as supplied includes a perpetual firmware licence for the operating system.

The body of the MultiPrep CellSprint contains a multi-layer extraction filter for the extraction of vapours. This filter is a self-contained unit with a HEPA(High Efficiency Particulate Air) filter and activated charcoal to prevent particulate matter and hazardous fumes escaping into the laboratory space. If optional heater-fan is fitted, samples can be warmed to 37ºC inside the instrument.


© Genial Genetic Solutions 2013 1

Features

A - Safety1. The unit is supported at four corners by castors to prevent tipping while moving. Each castor can be locked for parking.

2. The Main door has a safety interlock switch. It cannot be opened by accident. All moving parts are immobilized when this interlock is broken. This makes loading the unit safe, but also ensures safety when interrupting a harvest.

3. There is a clearly-marked Emergency Stop (ESTOP) button on the exterior of the machine which similarly cuts all power to any moving parts.

4. There is also a clearly marked Reset button for re-enabling the system after ESTOP or closing the door (interlock).

5. There is a clearly-marked power isolator switch which removes power to all parts of the unit.

6. The HEPA filter includes an activated charcoal layer so it is effective against noxious gas as well as organic particles. It is in continuous operation to maintain the air quality experienced by the operators.

7. Samples can be loaded away from the machine in a loading tray. Each operator is thus allowed to load the tray at whatever working height he or she finds comfortable, preferably in a laminar flow cabinet (hood).

8. The centrifuge only runs when the centrifuge cover is in place. The cover can easily be lifted to access the centrifuge bowl for cleaning. This item is easily within reach of the operator.

9. In normal operation, an alert is given if ever the centrifuge begins to become unbalanced. In such case the centrifuge is automatically and gently brought to rest.

10. The heaviest items dealt with by the operator (the reagent and waste vessels) are kept low - almost at floor level - to facilitate lifting.

11. The hypotonic delivery system has fluid gland packing. This ensures that the sealing between valves, pumps, and the reagents they control remains sound. They are designed to cool components and to take up any wear automatically and ensure leak-free operation. (The fluid system employed here uses only distilled water. It is sealed and normally requires changing twice a year.)

12. The exhaust line of the vacuum pump is connected in series to two small bottles, the first of which acts as a moisture trap. The second contains scrubber granules which remove noxious gases.

13. Where power cables and fluid lines are subject to movement (as seen here with the hypotonic lines on the right), these have been encased in a flexible shield to prevent chafing and snagging.



B - Sample Security1. Each sample has a unique place in the loading sequence and it can be tracked if necessary throughout processing.

2. Each sample is contained in a chamber separate from other samples. The sample is automatically sealed at all times except for the addition of reagent or the aspiration of supernatant.

3. The sample tubes are completely enclosed in strong, tough centrifuge buckets to guard against hazard should a sample tube break.

4. Once sealed, these buckets are proof against cross contamination of samples even if one should be accidentally knocked over on the bench.

5. Sample tubes can be loaded to a loading tray (caddy) away from the unit. This means that samples can be kept within a prescribed temperature environment until the moment when harvesting should begin, also

6. Samples can be loaded in convenient proximity to a barcode reader or sample storage facility.

7. The loading tray or caddy is designed to support each bucket securely at top and bottom while loading samples, delivering reagents or aspirating supernatant, to prevent spills.

8. Concerning the delivery of reagents, protocols can be managed to adjust the rate of delivery and pipette tip height to maximise re-suspension yet avoid splashing. Aspiration pipettes are washed in fresh reagent before moving from one sample tube to another.

9. The system is designed so that no pipette ever travels over the open mouth of a sample tube other than the one it has to address.

10. The effectiveness of fixative deteriorates over time, so fresh fixative is prepared immediately before it is used (if optional feature is fitted). The container for this fixative is held within an insulated box which has space for optional cooling gel packs to be packed around it; this also serves to preserve the mixture’s effectiveness. Before fresh fixative is prepared, the old mixture is automatically evacuated from the vessel. Delivery lines and pipettes are purged of old solution and primed with fresh fixative immediately before delivery.



C - Operation1. Aspiration is made as effective and as quick as possible. The steps towards achieving this efficiency are discussed within

the “Aspiration Notes” section.

2. (If optional feature is fitted) Fresh fixative is made each time at the point when it is needed. How old fixative is purged from storage and the lines primed with fresh is discussed in the “Fix Mix Bottle” section.

3. Ceramic piston pumps are used for most fluid movement. These are reliable, hard wearing, and maintenance-free de-vices. They deliver precisely metered pulses of liquids. Where a near-constant stream is required, two pumps are em-ployed to the same end with a respective phase difference of 180 degrees. Extraction of waste or aspiration of super-natant is done exclusively using vacuum (negative pressure) or gravity plus vacuum pressure. This isolates mechanical pumps from indeterminate materials such as bone flecks in a tissue sample.

4. Cellsprint is constructed using materials and coatings which are resistant to the chemicals, temperatures and stresses repeatedly encountered during automatic operation. There is extensive use of stainless steel for key components (such as the springs in the centrifuge buckets). Other metals are heavily anodized or powder-coated. All polymers are rated for prolonged exposure to the specific reagents employed in harvesting.

5. All pipettes are fashioned from teated stainless steel to minimise fluid adhesion. Cellsprint uses routines which minimise the immersion of probes in solutions; this simplifies and accelerates the process of washing the probes between sample tubes.



Aspiration Notes

Liquid SensingCellSprint has an automated routine to make the aspiration of supernatant as efficient as possible. Probes1 work in pairs within each sample tube. The solutions used for harvesting are all electrolytes so when a pair of probes touches the liquid, a electrical circuit is closed and the presence of liquid is detected. In normal operation probes descend until the solution's surface is detected. Aspiration through one probe proceeds from this point while the pair continues to descend for 20mm. The probes then pause their descent. If the sensing circuit is broken, then aspiration so far has been successful, and the probes descend another 20 mm before pausing again to check that the tips are clear of liquid. This process continues until the required depth of supernatant has been aspirated.

At the target depth the probes are slightly withdrawn and if liquid is still detected, this suggests that the aspirating probe is blocked. In this case aspiration is automatically retried but this time using the other probe. If during the withdrawal phase liquid is still detected, the pipettes are moved to the wash station for unblocking before aspiration is attempted again. Should the withdrawal phase again detect the presence of liquid, aspiration stops and an alarm is sounded to alert the operator, and a message is sent to the HMI. See Appendix A.

Collision SensingIf, for example, a sample tube has been wrongly inserted into a centrifuge bucket, there is the possibility of the probe tip meeting a solid object during its descent. To prevent damage to the probe, there is a microswitch within the pipette head assembly which stops downwards travel immediately when it is triggered. The same sensor protects the operation of the Bucket Opening mechanism. This minimises the risk of the pipette assemblies causing damage to themselves or anything which they might encounter.



1 Please note the terms probe, tube, and pipette tip are used interchangeably.

Fix Mix Bottle (OPTIONAL FEATURE)Fresh fixative is prepared immediately at the beginning of each phase in which it is to be used. This mixing is done in the Fixative Vessel (or fix-mix bottle). This resides behind the reagent scales and isn’t monitored by scales as it is prepared to measure and almost completely emptied before preparing fresh fixative.

There are normally three tubes inserted through the cap to this vessel.

• The loading tube (blue) alternately pumps acid and methanol in the proportions required for the Fixative concentration set in the phase parameters. This is normally a clear, plastic tube.

• The evacuation tube (straight end) draws away and discards unused fixative into the waste vessel. It is made of stainless steel and it reaches the top of the convex bottom of the vessel, avoiding completely emptying it.

• The delivery tube (curved end) feeds the dispensing pipettes. This is also made from stainless steel and curls into the lowest part of the bottle so that it is always dipped in fixative and no air can get into the lines.

Notes

1. Having the two reagents emerge from the single feed pipe encourages rapid and thorough mixing in the preparation of fixative.

2. The evacuation tube draws away unused fixative until there is no more left at the mouth of the tube. The distance of this mouth from the lowest point of the vessel defines the amount of residual fixative. This residual amount remains behind to prevent the ingress of air to the delivery tubes, preserving the efficiency of the pumps and obviating the need for priming each time. However, as soon as fresh fix is prepared, the old fix is purged and the delivery lines are primed with fresh solution.

3. The evacuation tube is wired via a logic controller or CPU (which commands a valve-actuator) to the delivery tubes. While there is fixative solution between the evacuation and delivery tubes, the circuit is closed and the valve permits vacuum extraction. Once this circuit is broken, the valve closes shortly after and fresh fixative is prepared. The re-establishment of this circuit is also used as a signal to the controller that it is safe to begin purging the delivery lines of old fixative.



Difference between Resuspension and DeliveryDuring Resuspension, reagent is injected alternately or simultaneously out of both probes in order to break up the pellet(*). This, typically, is delivered at a faster rate than normal delivery of reagent. If the total volume delivered is excessive, the probes may become immersed in the resuspended sample solution. This can be fully avoided by reducing the “Resuspension Height”.

Unlike Resuspension, reagent Delivery may only utilize one probe, and is usually delivered at a lower rate than that used for Resuspension. The purpose of this delivery is to bring the total tube volume up to the required amount and maximise the exposure of the sample to the reagent(*). Consequently, the probes should deliver reagent from outside the tube. Total volume should not exceed tube capacity.

Delivery Volumes • Hypotonic resuspension/delivery is in multiples of 0.7ml and is automatic. If you input a value that is not a multiple of

0.7ml, the unit will automatically round to the nearest correct value.

• Fix resuspension/delivery is in multiples of 0.7ml and is automatic. If you input a value that is not a multiple of 0.7ml, the unit will automatically round to the nearest correct value.

Delivery and Resuspension Dispensing Rates The maximum suggested rate for Fixative delivery is 1.0 ml/s, whilst the maximum recommended rate for Hypotonic delivery is 1.3ml/s.

*Extensive trials have shown the following methods (which are used by CellSprint) to be the most effective:



1. Rapid parallel de-livery causes turbu-lence, resuspending the pellet.

2. Long, steady pulses down one side of the tube encour-ages deep mixing.

Parts Nomenclature

A - ExteriorA1 Main Door[also: Loading door, sliding door]

A2 Reagent Doors

A3 Pump Chamber

A4 Locking Castors

A5 Power lead (See Appendix B)

A6 Filtration system (Also see “Periodic Maintenance”)

A7 Control panel

A4 - CastorsA4.1 Castor

A4.2 Brake Foot

A4.3 Knurled Wheel [Adjuster]

There are four self-steering Castors on the CellSprint Base. Each is “braked” by turning the knurled wheel (A6.3) anti-clockwise to lower the Brake Foot. These units are designed to have the CellSprint resist the lateral movements of opening doors, extending the Bottle tray etc. They are not meant to level the unit. Finger pressure should be sufficient to lock or release (by turning clockwise). The rear castors are within arm’s reach so finger pressure is all that can or should be applied.

A6 - Heating & Air FiltrationA6.1 Heater/Blower unit (OPTIONAL)

A6.2 External Vent

A6.3 Space Wall Duct

A6.4 Process Chamber Inlet

A6.5 Centrifuge Exit Vent (flexible ducting pipe removed for clarity)

C4 Filtration Unit

A6.6 Filtration Unit Inlet

A6.7 Filtration Unit Outlet

C3 Wash Waste Stand



A2

A3

A4

A1

A7

A4.1

A4.2

A4.3

A6.1

A6.2

A6.3

A6.6

C4

A6

A fan heater A6.1 (OPTIONAL FEATURE) blows warm air into the Process Chamber. This is removed via the Centrifuge Exit Vent A6.5 into the top of the filtration unit A6.6. Filtered air is blown through a flexible duct (not shown) under the Wash Waste Stand to the vertical Space Wall Duct and back to the heating unit conserving heat. Imbalances in air flow are relieved through the external vent A6.2.

The lid of the Filtration Unit can be removed from within the Reagent Chamber (see Periodic Maintenance). The filtration unit itself is not fastened to the floor, so it can be moved towards the door for easier access.

A7 - Control PanelThe Control Panel refers to the area to the right of the main loading door. It includes:

A7.1 Reset Button

A7.2 ESTOP Button

A7.3 HMI (Human Machine Interface - Touchscreen)

A7.4 Isolator Switch

A7.5 USB Port

The ESTOP button is the Emergency Stop button and removes power from all mechanical movement and processing steps in the machine, enabling the door to be opened and the operator to interact with the system’s internal parts. (Opening the door achieves the same effect.) The Reset Button re-enables the moving parts if they have been stopped either by the door opening or by the ESTOP button. The HMI is the Human Machine Interface. This is a touch-screen panel which is used for all details of protocols. Please see the sections on Setup, Regular Processing, and Protocols. The Isolator Switch removes power from the unit as a whole, rendering it safe for the replacement or maintenance of components.

The USB port is used to load the control program and updates. It should not be accessed by anyone other than Genial Genetic Solutions Personnel without the express guidance from an application specialist.



A6.4

A6.5

A6.6

C4 C3

A6.7

A7.2A7.1

A7.3

A7.4

A7.5

B. Main Processing ChamberB1 Carousel [caddy, loading tray] (in 48 place models only)

B2. Turntable [centrifuge cover or lid]

B3 Centrifuge

B4 Wash Stations

B4.1 Reagents Washstation

B4.2 Aspirate Washstation

B5 Towers

B5.1 Gripper (in 48 place models only)

B5.2 Aspiration

B5.3 Hypotonic

B5.4 Fixative

B1 - Carousel (in 48 place models only)B1.1 Carousel bucket bearings

B1.2 Carousel upper ring

B1.3 Carousel pillars

B1.4 Carousel Lower ring

B1.5 Carousel floor



B5.1

B5.2B5.3

B4.1

B4.2

B1

B2

B5.4

B1.1

B1.3

B1.4

B1.5

B1.2

B2 Turntable/Centrifuge Cover (in 48 place models only)B2.1 Turntable drive motor

B2.2 Lifting Recess

B2.3 Centrifuge Cover Securing Screws

This illustration shows how centrifuge buckets fit on the carousel and how the carousel is fitted to the turntable. The turntable also serves as a cover to the centrifuge. The centrifuge cover is hinged on its far side. When slid fully to the left, and with the carousel removed, the lid can be raised (see “Periodic Maintenance”)

B3 CentrifugeB3.1 Centrifuge Bowl (not shown)

B3.2 Sealing Dome (not shown)

B3.3 Rotor

B3.4 Rotor Bucket Pins

B3.5 Rotor Anchor Screw

B3.6 Centrifuge Bucket

B3.6.1 Centrifuge Bucket Lid

B3.6.1.1 Flaps

B3.6.1.2 Spring

B3.6.1.3 Pin

B3.6.2 Centrifuge Bucket Gasket

The rotor is designed to cope with the stresses of high speed centrifuging. Scratches can detract from the strength of this unit so it should be inspected regularly. (See “Periodic Maintenance”.)

The Bucket Top is supplied as a whole and it isn’t necessary to separate the parts for routine cleaning. Over time and with repeated washing, the flaps might get sticky and not spring shut automatically. They have been designed such that it doesn’t require skill or special tools to dismantle them for thorough cleaning or replacement if ever necessary.



B2.1

B2.3

B2.3B2.2

B3.5

B3.3

B3.4 B3.6

B3.6

B3.6.1

B3.6.2

B3.6.1.1 Flaps

B3.6.1.2 Spring

B3.6.1.3 PinThe flaps have bungs to resist spills. There are internal walls to completely isolate each sample. The gaskets fit snugly in recesses of the bucket base. Even if a sample tube should fail, the spill is

contained in its chamber.

B5.1 Gripper Tower Assembly (in 48 place models only)B5.1.1 Housing

B5.1.2 Bucket Sensor

B5.1.3 Gripper Arm

B5.1.4 Gripper Eye

The Gripper Eyes should fully engage with the bucket lugs (circled, below). If either lug is damaged then the bucket should be replaced.



B3.6.1.2B3.6.1.2

B3.6.1.3

B3.6.1

B3.6.1.1

B3.6.2

B3.6

Correct pick-up. Both lugs en-gaged, and firm contact with the sensor. confirms presence of bucket.

Failed pick-up. Sensor fails to detect presence of bucket.

B5.1.1

B5.1.2

B5.1.3

B5.1.4

B5.2, B5.3, B5.4: Dispensing TowersThe Assemblies for the Aspiration, Fixative Dispensing and Hypotonic Dispensing Towers are largely identical. (The aspirating pipettes have a wider bore, and deliver a negative pressure, however.)

B5.3.1 Pipette Head Assembly

B5.3.2 Pipette Head Retaining Nut

B5.3.3 Pipette (pair) [also: “Probes”. (Two tubes pared to reveal interior sleeves.)]

B5.3.4 Bucket Opening Rod

B5.3.5 Bucket Opening Fork [not all detail shown here]

When replacing damaged pipettes, first note the position of the original before loosening the Retaining Nut.



B5.3.1

B5.3.2

B5.3.3

B5.3.4

B5.3.5

B4.1, B4.2 Wash StationsThere are two Wash stations, the one nearer the main loading door being designated the “reagents” Wash station. Wash stations contain four separate wells or weirs for washing the probe ends. These are plumbed to a peristaltic pump which evacuates the waste after each rinse or prime. Each well has an indivdual feed tube near the top, which can carry hypotonic or methanol according to the phase in the harvest. (For clarity's sake, the fluid lines themselves are not shown in this illustration.) These items are firmly fixed and have no moving parts.



C. Reagents ChamberC1 Bottle Drawer

C2 Bottle Cage

C3 Wash waste stand

C4 Air Filter Unit

C5 Fixative Perch

C6 Fix Perch Lid

Pressure plates:C7 Vacuum Bottle [Aspirate Waste, Aspirated Supernatant]

C8 Wash Waste

C9 Hypotonic

C10 Acetic Acid

C11 Methanol

Note that Pressure Plates (weigh scales) can be optionally lifted. They should be installed in their prescribed positions as far as possible. If they are moved to accommodate a differently-sized vessel, they should be placed so that they do not overhang an edge nor touch the cage or another pressure plate.

The filtration unit (C4) is not fixed to the floor so it can be moved close to the front whenever the filter cartridge needs to be replaced. The unit is connected to the left wall by concertina ducting. There is enough free play in this to allow for gentle movement, so this unit can be moved towards the front for easier access.



C6

C5

C4

C3

C8

C7 C9C10 C11

C1

C2

Reagents Chamber VesselsFluid lines to and from reagent vessels are supplied pre-assembled. A user need only replace reagent bottles or the waste (vacuum) bottle after emptying. The tubing between the cap and the valves and pumps does not need to be disturbed except in the case of a replacement. If the replacement reagent vessel differs in size from the original then the length of tub intruding into the vessel may need to be adjusted. The tubes are a push fit in the caps.

Above are the three vessel caps supplied as standard. 1. has an outlet and a vent used for Acetic Acid and Methanol Vessels. 2. has an inlet and an outlet and is used for the distilled water “fluid gland” system (See “Periodic Maintenance”). 3. has three outlets and one inlet. This is the cap used for the Fix/Mix bottle. The caps conform to the GL45 standard and will fit a wide variety of vessels. Illustrated right is this cap on 2.5, 2.0, 1.0 and 0.1 litre vessels.

Where a feed pipe’s end must be immersed in the fluid, there is a lightweight filter screwed to its end. (See illustration, right.) The tube should be pushed down so that this filter is as close to the bottom as is practicable. If a different sized vessel is used as a replacement, this adjustment should be attended to immediately. The pipe’s union with the cap is a push fit so this can be achieved quite simply.

Aspirant Waste Vessel [“Vacuum Bottle”] This has two inlets and a vent to an air pump to create a partial vacuum. When full this can be the heaviest vessel used; it has two handles for easy lifting at the shoulders. Note that the lid must be seated properly to ensure a vacuum.

A 3-way manual escape valve is installed over the vacuum bottle lid to allow air into the bottle when the lid is too tight to turn. Care must be taken to return the valve to the default closed position (with the OFF handle pointing at the open port) in order for the system to generate enough vacuum to aspirate effectively. As a matter of good practice, turn the handle 1/4 turn anticlockwise when letting air in, so that the vacuum gauge reads 0 and acts as a reminder that the valve must be closed.



2.1. 3.

Sometimes, to investigate the location of a blockage, lines might be exchanged until the offending item is identified. Use the schematic diagram below as guide to this process.

Fix Mix vessel cap showing the electrical connectors (If option is fitted). See the section on the “Fix Mix Bottle”. The image below shows the relationship between the vessels in the Reagents Chamber.

Schematic of fluid linkages



Initial Checks APositioning, physical access, proximity to reagents, benching, power access, UPS

Although compact, the CellSprint is a substantial unit weighing in over 400 kg. It requires a strong, stable, level2 floor space. Solid floors are therefore to be preferred to floor-boarding. Once in place it should not be moved needlessly, so access to power should be arranged before siting. (See Appendix B for power requirements and UPS provision.) In all other respects the CellSprint is self-contained and needs no provision for drainage or additional ventilation.

In normal operation it is accessed only from the front so it can be mounted against a wall, leaving a 10cm gap at rear. Periodic maintenance and inspection requires access to the side panel on the right. This needs a clearance of 62 cm (about 2ft) which is the room required for a person to stand, turn and bend. Such maintenance is a rare event, and since the CellSprint can be maneuvered very easily, it can be placed next to another unit, bench or even a wall.

Loading operations at the front involve tissue samples and bottles for reagents and waste, so ideally these areas should not impinge upon any main thoroughfares in the laboratory.

Reagents used by the CellSprint are typically loaded in swapped-out standard-sized flasks without a need for decanting. Some extra space may be required while changing these. (Having these items stored fairly close to the unit would be advantageous.)



2 There are adjusting screws at each corner castor, but these are to inhibit lateral movement only. The CellSprint is fairly tol-erant of small deviations from the absolute level, but if the unit is on the point of rolling in any direction, consider re-siting!

1. Minimum installation space dimensions. Note that the power cable is plugged in at the rear of the machine. The back panel should be no nearer a wall than about 100mm.

2. Working space required. The main door needs to be lifted only about 30 cm (1 ft) to allow loading of samples.

3. Changed dimensions when doors are fully opened. Note that the main door lifts up 50 cm to allow access for cleaning. The side door is only opened for servicing/replacement of pumps and so can be discounted for everyday usage.



754 mm1200 mm

1870 mm

2370 mm

1820 mm

1215 mm

2170 mm

Initial Checks BThese are the checks which should be made before commencing harvesting, ideally at the start of the day:

1. Push the corners of the unit to see if there is any lateral or back-and-forth movement. The machine is anchored by four floor stops at the corner castors. Temperature and vibration over time may cause these to ease their grip. If the machine can be moved at all then these should be tightened. (See Castors)

2. Check that the main door lifts easily and closes completely. If there is a problem, this should be dealt with before samples are offered up to the machine.

3. Inspect the interior of the main processing chamber to check for leaks and/or spills. If cleaning has recently taken place, check that the centrifuge cover has been screwed back in place and that no cleaning materials (cloths, bottles) have been left in the centrifuge bowl or main Processing Chamber.

4. Visually inspect the pipettes in the towers. Look for any obvious deviation from the normal configuration of pipettes. These should all be regularly spaced, parallel and perfectly level with each other.

5. While there, listen for the sound of the extractor fan for the HEPA filter unit. This should always be running when the machine is in use. In most cases when the filter approaches saturation there is a characteristic odour from the acetic acid vapour which becomes perceptible long before it becomes hazardous so a visual inspection is seldom required.

6. Look at the tubes to the reagent and waste bottles to check that they have not been disturbed when the vessels were last replaced. Look (and smell) for leaks, and for the correct mounting depth in each. See the notes on Waste Vessels. Check that there is sufficient reagent and capacity in the waste bottles for at least the first harvest.



SetupThe Setup utility can be accessed from the Welcome screen of the HMI. There are four configuration items, Scales, Heater, Protocols and P&P (Pick & Place). They can only be accessed before starting a cycle and are only relevant if the optional feature has been fitted. You can use this utility to examine the settings. However, any changes to existing values require a password. There is a low security password (normally 1111) for protocol changes and a high security password (normally 4444) for engineering parameters.

Pressing the Scales control produces this console:

MMMM shows the state of the scales. These are the metal pressure plates located in the Reagent Chamber. These can be turned on or off using the large Mode-/Mode+ buttons at the bottom of the display. The display shows the volume contained (or available) for each vessel. This volume is calculated from known densities and the mass of the reagent concerned. The mass of reagent is calculated once the weight of the vessel has been subtracted from the weight reported by the pressure plate.

Touching the double chevrons causes the MM in the display to itself become a double chevron. If this is now

touched, the weight in grams of a particular empty vessel can be declared by placing the vessel on the pressure pad and pressing Teach. This captures the current signal from the pressure pad to define the Bottle weight in grams. When done with this screen, press Back. (Note that this need only be done once, and needs redoing only if the standard bottle is changed for a different type.)

Pressing the Heater control delivers this console. This is straightforward enough to use. The switch is either on or off and this is reported in the upper right [MMMM].

(Note the reminder that the fan is always on. This minimises undesirable vapours in the unit and helps control temperature more accurately. As most of the time, vapour-free air is passed through the HEPA filter, it does not clog any faster) that it would if there was intermittent operation.

By default, the set temperature is 37˚c. The Status message reports either “Not at temperature” or “Temperature reached”.

Pressing P&P (in 48 place models only) allows to disable the Pick and Place feature so that up to 6 buckets can be directly placed in the centrifuge.



Protocols: Description, ManagementUp to 16 protocols - each of up to 9 phases - can be stored by the CellSprint and each can be edited as required. One protocol may be supplied for blood and one for bone marrow as exemplars and for testing. There may also be a test protocol for commissioning and diagnostic purposes.

Delivery, Resuspension, Pellet DepthReagents are dispensed from probes whose tip is described as being “x” millimetres from their home or datum position at the top and travelling vertically down. As “x” increases, the probe descends further towards or into the sample tube. Delivery and Resuspension parameters therefore include a “from x mm” parameter.

In the interest of saving reagents and time, the dispensing probes aren’t normally washed between operations. To avoid cross contamination, the dispensing heights must be set such that the probes aren’t dipped in or splashed by the sample.

The aspiration of supernatant is similarly specified in terms of depth. “Pellet depth” is a length expressed in millimetres and should be understood as the distance the aspirating probe travels into the test tube from the datum or rest position. As the value of “pellet depth” increases, the volume of the liquid left in the test tube decreases.

ParametersFor every phase, 14 parameters must be defined:

• Phase Name

• Spin Time

• Spin speed in RPM (max. is 1500 rpm but recommended limit is 1250 rpm)

• Pellet depth in mm (higher number = less supernatant)

• Resuspension Reagent (either hypotonic of fixative)

• Resuspension Height in mm (position from which the reagent is delivered; higher number = further in the tube)

• Resuspension Volume in ml

• Resuspension Rate of delivery in ml/sec

• Delivery Reagent (either hypotonic or fixative)

• Delivery Height in mm (position from which the reagent is delivered, higher number = further in the tube)

• Delivery Volume in ml

• Delivery rate in ml/sec

• Fix Ratio (disregard for hypo phases)

• Incubation time (seconds)

Relationship between incubation and spin timesThe instrument calculates the timings for other activities around the incubation time declared in seconds by the user. If the incubation time is shorter than the spin time in the corresponding phase, the spin time takes precedence over the incubation time.



A note on editing protocols To make changes to the phases in a protocol (as opposed to inspecting the details of it) requires a password to be entered. To do this, press and hold any unused portion of the screen for 5 seconds. This will call a login screen (pictured below). Use the arrows to navigate to “PASSWORD”, and press the enter button. Enter the password and press enter button. You will be taken back to the window you were viewing, and now all of the parameters become ‘clickable’. Pressing your finger over the value you want to change calls the Numeric Input Screen similar to the one shown below.

Use the scroll arrows to highlight the PASSWORD option. Then press the Enter key at bottom right.

This screenshot shows the PASSWORD prompt that requires the input of a numerical password.

Touching regions marked in RED on the above display (or any similar feature on other pages) calls the input console featured below:



Managing ProtocolsFrom the Welcome screen, press Setup, then Protocols to reveal this screen:

Pressing Protocols delivers this console. “Which protocol do you want to change?” here should also be read as “What number will you give your new protocol?”

From this point an existing protocol can be copied to this position by touching Copy From. Individual phases of a protocol may be edited by pressing Phases. The Global control sets conditions for all phases in this protocol.

There can be up to 9 phases in a protocol. The double chevrons move the display to the next or previous phase while Exit saves the changes and returns the user to the previous screen.

The may be any of the following:

• DISABLED• HYPOTONIC 1• HYPOTONIC 2• PRE-FIXATIVE 1• PRE-FIXATIVE 2• FIXATIVE 1• FIXATIVE 2• FIXATIVE 3• FIXATIVE 4• SPINDOWN

These names are for user reference; the system program operates on phase numbers. However, any phase that is named DISABLED will be ignored and skipped by the program. By this same token, all unused phases after the SPINDOWN must be set to DISABLED when a new protocol is entered.



The parameters for the protocol phase should be understood as follows:Spin: X seconds of centrifuging at Y revolutions per minute Pellet depth: This is the amount of residue not aspirated after centrifuging. This is determined by having the aspirate probe tip descend to P mm from the datum (rest) position. This can be quite large in comparison to the few mm of actual residue which is considered the pellet; the larger this value, the smaller the pellet.Resuspension and Delivery: These are specified the same way. The Reagent type can be either Hypotonic or Fixative. This is injected from a probe whose tip is Z mm from the datum (rest) position. A volume of V millilitres is delivered at a rate of R mls per second.A step with SPIN TIME, PELLET DEPTH,RESUSPENSION VOLUME or DELIVERY VOLUME left as zeros or nulls will be ignored by CellSprint.Fix Ratio can be any of the following(methanol:acetic acid) 2:1, 5:2, 3:1, 7:2, 4:1.These values are selected by scrolling with the UP/DOWN arrows rather than inputting values in the numeric keys.

Fresh fixative is prepared at each phase where it is required so the ratio can be varied from phase to phase.

Global Settings are used to configure the Centrifuge behaviour. Setting the approach to and departure from the maximum revolution speed helps control the appearance of vortices in the tube which might disturb the pellet.

The initial incubation time can be used to countdown the colcemid exposure time. Samples can be loaded after adding colcemid and the system will start the harvest automatically when the declared incubation period lapses.

Exit returns the user to the previous screen.



Loading Samples1.Insert the sample tubes (with their lids off) into the centrifuge buckets. The design for the internal bottom of these buckets will accommodate most hemispherical and conical ended tubes between 12 ml and 15 ml capacity. The bucket base has numbered stations. This means that tubes can be tracked in their unique positions in the harvest. The gasket (here shown in blue) comes in two sizes to accommodate test tubes of different diameters. It is removable for cleaning.

2.The lid of the centrifuge bucket is kept in place with two side clips. By sliding thumb and index finger down either side of the cap, the clips can be pushed open to release it. The arrow on the cap and the triangle on one of the base lugs should be aligned to avoid cross-contamination of samples if the lid is removed and replaced.

3.The convention is that the arrow on the ridge should point away from the centre of the rotor (and carousel in 48 place models).

Loading the carousel (48 place models only)

Buckets should be placed into the carousel so that the pattern is always symmetrical. If any odd number of buckets is to be processed, add a bucket filled with empty sample tubes on the opposite side of the carousel to preserve the symmetry. Buckets should be loaded in the following order: 1 & 7, 2 & 8, 3 & 9, 4 &10, 5 & 11, 6 & 12, using the station order shown below.

Shown here are buckets loaded at 1, 2, 7, 8. Number 1 is at the “point”. 2 is depicted in green.



1

2

3

6

4

7

5

8

9

10

11

12

Loading the centrifuge rotor (24 place models and manual mode)

Buckets should be placed into the centrifuge rotor so that the pattern is always symmetrical. If any odd number of buckets is to be processed, add a bucket filled with empty

sample tubes on the opposite side of the rotor to preserve the symmetry. Buckets should be loaded in the following order: 1 & 4, 2 & 5, 3 & 6, as shown.

Balancing the centrifugeThe system processes test tubes in multiples of 8. Test tubes containing an equal amount of water as the sample volume should be placed in the buckets to make up for these numbers. It is possible to place empty test tubes in opposing buckets as long as the centrifuge weight is balanced. Though it is possible to leave empty spaces in the buckets as they are impervious to the reagents, it is recommended to fill every slot to keep the buckets clean and dry.



Regular Operations ACellSprint provides a genuine load-and-walk-away mode of operation. However, regular operation involves a shortlist of processes and checks to be observed. A short, single page “Bench Document” appears in the next section.

Making Checks1. Inspect the interior of the main processing chamber to

check for leaks and/or spills. If cleaning has recently taken place, check that the centrifuge cover has been screwed back in place and that no cleaning materials (cloths, bottles) have been left in the centrifuge bowl or main Processing Chamber.

2. Visually inspect the pipettes in the towers. Look for any obvious deviation from the normal configuration of pipettes. These should all be regularly spaced, parallel and perfectly level with each other.

3. While there, listen for the sound of the extractor fan for the HEPA filter unit. This should always be running when the machine is in use. Some units have crystals which change colour when they require changing. In most cases when the filter approaches saturation there is a characteristic odour from the acetic acid vapour which becomes perceptible long before it becomes hazardous so a visual inspection is seldom required.

4. Look at the tubes to the reagent and waste bottles to check that they have not been disturbed when the vessels were last replaced. Look (and smell) for leaks, and for the correct mounting depth in each. Check that there is sufficient reagent and capacity in the waste bottles.

5. If you have not already done so, make sure now that the main loading door is closed.

Declare Number of Buckets to be processedThis is done using the HMI. Click Run from the Welcome screen.

Next, press the number of buckets to be processed in this harvest. There must always be an even number of buckets regardless of the number of samples. If necessary, fill buckets with empty sample tubes to ensure a balanced pattern of buckets. (See “Loading Samples”). From the bucket information, the system can begin to calculate reagent requirements and know where the samples should be located.



Choose a protocolAs with the previous screens, a single touch is all that is required here. pressing the protocol number calls the next screen. (Note that the Setup utilities can be accessed from here if at this point you realise that you need to amend or create a protocol or other setting. See the instructions regarding access passwords in “A note on editing protocols”)

The console illustrated right appears. From this, processing can be paused or reset altogether. The processing stages should proceed automatically from this point with no further intervention. Optionally, each process can be stepped through manually, and its progress inspected in detail. This might be done to complete a “stalled” protocol which cannot restart automatically. For guidance about interruptions to processing or Manual Control in general, see page 36.

From this point the messages in the Main Commentary Report should be followed

Datuming OperationPress the Blue Reset button to enable all the motor drives. Then press OK when the prompt “PAUSED - press OK to resume” appears. A position recovery routine will happen if the system has run other cycles before power cycling. This takes about 20 seconds. Otherwise, the prompt “Press OK to datum machine” will immediately appear. By pressing OK, the system will do a complete homing routine of all moving parts, prime the hypotonic lines and rinse the aspiration probes. When complete, the user is prompted to “Load samples or press OK if loaded”. If the samples haven’t yet been loaded, the Blue Reset button must be pressed after closing the door, followed by pressing OK when the message “PAUSED - press OK to resume” comes up. A position recovery routine will take place.

Following the prompt “Press OK to run cycle” will start the harvest cycle.

Monitor Progress, or walk awayThe protocol in use appears in a static display at the top, but there are also three “dashboard” displays showing the Current Centrifuge Speed (in revolutions per minute), the time before the centrifuge finishes its current spin and, if an incubation period has been specified, the time to finish this. If anything happens to interrupt normal processing, an audible alert sounds and and error is displayed on screen.

Note that there are 5 “narrative’ displays and 3 icons:

1. Bucket Geometry Messages

2. Protocol Number

3. Main Cycle Messages

4. Sub-Cycle Report Messages

5. Main Commentary Report

6. Heater ON or OFF (crossed out)

7. Automatic Pick & Place ON or OFF (crossed out) (for 48 place systems)

8. Scales ON or OFF (crossed out)



Main commentary report and instructions

Bucket Geometry 3

Sub-cycle status messagesMain Cycle status messages

0 0 0

Bucket GeometryThese reflect the initial loading of the buckets according to the 12 position carousel. In dual mode (running more than 6 buckets) this may not be apparent from looking through the door window, since some might be in the lower compartment, therefore this reminder is provided. (Note the symmetrical loading, from the diagrams on pages 26 and 27.)

Main Cycle MessagesThese show which set of buckets in the whole loading are being operated upon at any point. When processing up to 6 buckets, the reported status refers to the whole batch so no suffix is shown. However, when processing more than 6, the load is split into two batches (odd and even buckets), and the status has a suffix of either “1st Batch” or “2nd Batch”. What is being done with them is detailed in Sub Cycle Status Messages.

Sub Cycle Status MessagesWhat appears in the Sub Cycle Status Messages message window depends upon the protocol being used. The message indicates which bucket is under each station, whether it is being ‘treated’ or not.



Bucket Geometry MessagesNo Buckets2 Buckets (1,7)4 Buckets (1-2,7-8)6 Buckets (1-3,7-9)8 Buckets (1-4,7-10)10 Buckets (1-5,7-11)12 Buckets (1-12)

Main Cycle Status MessagesDisabledHypotonic 1Hypotonic 2Pre-Fixative 1Pre-fixative 2Fixative 1Fixative 2Fixative 3Fixative 4SpindownUdeifinedInitialisation1st centrifugeFinalisation** CYCLE COMPLETE **

Sub Cycle Status MessagesInactiveAsp1 / --- / ---Asp2 / Hyp1 / ---Asp3 / Hyp2 / Fix1Asp4 / Hyp3 / Fix2Asp5 / Hyp4 / Fix3Asp6 / Hyp5 / Fix4 --- / Hyp6 / Fix5 --- / --- / Fix6DatumingUnloading centrifugeLoading centrifugeCentrifugingSwapping bucketsWaiting for centrifugeWaiting for fixativeWaiting for incubation

Main Commentary ReportThis is a quick reference of the current action being performed so it sometimes repeats information given in other report windows. It is also the place to receive notification of errors and instructions about what to do next.

An explanation of some of these errors appears in Appendix A. Note the difference between Routine, Warning and Error messages, all of which may appear in this window. Errors are accompanied by an audible alert (a buzzer will sound until it is acknowledged by pressing the OK button once). If the problem is resolved, the cycle can be resumed where it was paused by pressing the OK button for the second time.



Main Commentary ReportNo messagesPAUSED - press OK to resumePress OK to datum machineLoad samples or press OK if loadedPress OK to run cyclePlease wait...Press OK to open doorPress blue button to continueWARNING! Check vacuum bottleWARNING! Check waste bottleWARNING! Check methanol bottleWARNING! Check acetic bottleWARNING! Check hypotonic bottleERROR! VIBRATION - Check centrifugeP&P FAIL! - Put bkt in slot under gripWaiting for manual commandDatuming machineOpening front doorLoading centrifugeUnloading centrifugeIndexing centrifugeSpinning centrifugeIndexing carousel CWIndexing carousel CCWOpening centrifugeClosing centrifugeAspiratingWashing aspirationDelivering hypotonicResuspending with hypotonicDelivering fixativeResuspending with fixativeERROR! CHECK FOR LEAKS/SPILLAGESERROR! CENTRIFUGE FOLLOWING ERRORERROR! CHECK PICK/PLACE LIMIT SWITCHERROR! CHECK ASPIRATION LIMIT SWITCHERROR! CHECK HYPOTONIC LIMIT SWITCHERROR! CHECK FIXATIVE LIMIT SWITCHPreparing to release doorERROR! ASPIRATION CLOG - CHECK PROBES!Making fixativeWashing hypotonicPriming hypotonicWashing fixativePriming fixative

Main Commentary Report (cont)ERROR! PICK/PLACE SWING FAILUREERROR! ASPIRATION SWING FAILURE ERROR! HYPOTONIC SWING FAILUREERROR! FIXATIVE SWING FAILUREUnblocking aspiration pipettesSwinging P&P clockwiseSwinging P&P counter-clockwiseSwinging ASP clockwiseSwinging ASP counter-clockwiseSwinging HYP clockwiseSwinging HYP counter-clockwiseSwinging FIX clockwiseSwinging FIX counter-clockwiseERROR! ASPIRATION PROBES CRASHEDERROR! HYPOTONIC PROBES CRASHEDERROR! FIXATIVE PROBES CRASHEDDATUM - Waiting for PP tower (V)DATUM - Waiting for ASP tower (V)DATUM - Waiting for HYP tower (V)DATUM - Waiting for FIX tower (V)DATUM - Waiting for HYP tower (R)DATUM - Waiting for FIX tower (R)DATUM - Waiting for pump 1DATUM - Waiting for pump 2DATUM - Waiting for pump 3DATUM - Waiting for hypo primeDATUM - Waiting for aspiration washDATUM - Waiting for centrifuge doorDATUM - Waiting for carouselDATUM - Waiting for centrifugeDATUM - Waiting for PP tower (R)DATUM - Waiting for ASP tower (R)RECOVERY - Waiting for P&P tower (V)RECOVERY - Waiting for ASP tower (V)RECOVERY - Waiting for HYP tower (V)RECOVERY - Waiting for FIX tower (V)RECOVERY - Waiting for P&P tower (R)RECOVERY - Waiting for ASP tower (R)RECOVERY - Waiting for HYP tower (R)RECOVERY - Waiting for FIX tower (R)RECOVERY - Waiting for centrifugeRECOVERY - Waiting for ASP tower (V)RECOVERY - Waiting for HYP tower (V)RECOVERY - Waiting for FIX tower (V)

Regular operations B: Bench Document1. Empty waste bottles if necessary & check reagent levels

2. Check doors are closed

3. If necessary, turn power switch (isolator switch) to “on”

4. Press Run

5. Select number of buckets involved in this harvest

6. Select protocol to be used for this harvest

7. When prompted, press Blue Button to enable all systems (the door must be well shut for this to engage).

8. Press OK Button on touchscreen when the message "PAUSED- Press OK to resume" appears. If this message stays on or fails to appear, check door and try blue button again. Give the motor drives a few seconds to initialise.

9. Press OK to datum when the prompt appears and allow the system to initialise, prime the hypotonic lines and rinse the aspiration probes.

10.When prompted, open door & load buckets or insert loaded carousel (for 48 position model), close door & press Blue Button or just press OK if the carousel was loaded previously.

11. If the door was opened to load the carousel, the system will perform a position recovery routine in case any moving parts were disturbed. Press Ok once more when the message “OK to run cycle” appears.

12. When the messages “CYCLE COMPLETE” and "Press OK to open door" come up, press OK to refresh the program.

13. Unload the carousel/centrifuge and close the door. The system is programmed to prompt a new harvest with the same protocol and number of buckets once the Blue Button is pressed again.

14. At this point it is possible to start a new harvest or perform the end of day cleaning. For a different protocol or number of buckets, Press Reset Cycle, then OK to acknowledge the warning and return to step 1. It is also safe to switch the system off and on again

between runs to refresh it.

15. To rinse the lines at the end of the day, replace the hypotonic bottle with distilled water, and press OK to Datum. The system can be safely switched off when it prompts to load the carousel again.



Press blue button to continue

6 Buckets (1-3,7-9) 3

InactiveInitialisation

0 0 0

Manual ControlA note about “Datuming”. This is a form of elementary calibration or zeroing of the moving parts to ensure accuracy in the processing of samples. The travel of these parts is measured from a Datum or rest position of origination. If these are accurately registered at the start and checked periodically, then probes and arms will not miss their intended targets. A Datum operation winds these parts back to their secure rest positions, so, while essential, it's not very spectacular! It usually takes about twenty seconds.

Datuming is performed automatically at the start of every batch of processing. If there is any interruption to regular processing however and the main door has been opened or the emergency button pressed, datuming should be done again as a matter of course as the machine will not be aware of the positions of arms, probes, etc when it restarts. Datuming ensures that there is no chance of the moving parts colliding when processing resumes. It also “zeroes” pumps and primes delivery lines. Whereas datuming occurs automatically upon resuming a cycle after the blue button needed to be pressed, in manual mode it is necessary to datum manually. This is because during testing parameters with manual moves, it is possible to drive the probe heads or gripper head into an illegal position. If the position recovery were allowed to happen automatically, it would return to that same illegal state.


Bucket Geometry 3

Sub-cycle status messagesMain Cycle status messages

0 0 0

Resuming after pausing continues processing from where it left off; Reset Cycle begins processing from the start. For this reason, the following reminder is issued when it is selected. If the Cancel button is pressed, the cycle will not stop running.

Manual Control lets the operator ‘drive” the machine step by step through processing. This can be used to complete a stalled operation; it can also be used to investigate the effect of a change to a protocol or phase within a protocol when optimising or designing protocols.

Pressing Manual Control produces a screen like the one shown below.

Pressing OK then issues a reminder to datum. If the Blue Reset button has just been pressed, a few seconds must be allowed for the motor drives to initialise.



The datum can also be commanded from the first manual window to appear.

Pressing Exit returns the operator to the Main Processing screen above. The side arrows (<,>) scroll from page to page through the series below.

The Stop button will interrupt any task but generally allows the pumps to complete their dosage so care must be taken when stopping in mid-flow.

Each action involved in processing can be performed separately to check that it works as expected, or to calibrate the machine for different test tubes, or to complete a harvest step by step. Successive screens can be called or recalled using the chevron buttons.




3


3


3


3


3

The previous screen in the manual operation series is a console for adjusting the vertical travel of the four “arms” in the main processing chamber to their working height. The screen displays the vertical position of each tower, measured from the top home position and increasing as it plunges deeper into the test tubes. The up and down chevrons adjust the height measured for the top datum or rest position by the “Stepping Increment” each time3. After each adjustment, the operation can be checked manually to see the effectiveness of the change. This feature is mainly used for setup purposes, for example to establish the depth the dispense probes must reach for a particular protocol.

The last screen swings each tower Clockwise or Counter-Clockwise after driving it up to the safe height of the home position.

Notes on manual control:• Manual control should be exercised with caution as it allows the user to drive the system beyond its limits and damage

can be caused to its components.

• The parameters used for all manual moves are taken from the first phase of the protocol selected. No distinction is made of whether the phase is DISABLED or of which reagent is declared for the resuspension and delivery tasks.

• The only exception is the Make Fix function. For this it is necessary to declare FIXATIVE in one of the dispense steps of the first phase of the protocol in question.

• If the door is opened or the emergency button is pressed, the blue button will need to be pressed and though counterintuitive, the only available button to re-enable the system within that environment is Stop.

• If an alarm state is reached, pressing the Stop button silences the buzzer. If the Stop button is pressed for a second time, the task will resume and in many cases worsen the alarm condition. Pressing Done instead will clear the alarm but will leave the system in an uncertain state where it’s quite often best to switch off and back on again if there are doubts.



3 The numeric input console (see page 26) is automatically conjured when the digits to be changed in the “Stepping Incre-ment” are touched, so this incrementing value can be easily adjusted.

Periodic MaintenanceDailyAt the end of every day it is advisable to replace the hypotonic bottle with one that contains distilled water and run the datum of any protocol, as if starting a new harvest. This prevents the hypotonic and aspiration pumps, valves and lines from forming crystals. When the routine is complete and the system prompts to load the samples, it can simply be switched off.

WeeklyIf Cellsprint is in constant use, a deeper clean should be done once a week. This consists of replacing the hypotonic bottle with a lab safe solution of antibacterial cleaner and running protocol “W” with 2 buckets. The buckets should have empty test tubes to start with. The wash cycle initially circulates the cleaning solution through the hypotonic lines and into the test tubes. Subsequently, the aspiration probes empty the test tubes and use an unblocking routine, which back-flushes the aspiration lines and thoroughly rinses the interior of the valves, manifolds, tubing and probes. The hypotonic lines are then submerged in the test tubes filled with the cleaning solution and kept there for a prescribed amount of time. Finally, the aspiration/unblocking routine is repeated, emptying the test tubes and giving the aspiration lines an extra rinse. After this, the W protocol must be run again using distilled water this time to remove any foreign chemicals from the system.

If the machine will be left idle for some time, then the same cleaning process should be performed but the dilute cleaner should be left in the lines to avoid any growth.

Thanks to the sealed bucket system used, the system keeps itself generally clean but you can wipe the internal surfaces, using normal lab cleaning products. In 48 place models the centrifuge cover can be slid to the extreme left, and the two securing screws can be removed and retained. The cover is hinged at its back edge so it can be lifted for easy cleaning.

When replacing reagent vessels or emptying waste, mop up drips and spills immediately. Once clean, wipe the surfaces with a a cloth moistened with de-ionized water.

All accessible surfaces in the Reagent Chamber should be wiped with a cloth moistened with de-ionized water. Pressure pads can be lifted and replaced in turn for this. Ensure that they are replaced so as not to foul other pressure plates. Generally speaking, no cleaning of the

interior or exterior should be done with abrasives or heavy solvents but a number of lab safe cleaning agents are acceptable.

MonthlyEvery month the level of distilled water in the fluid packing gland reservoir should be replaced. The reservoir for this system is located on the floor of the Pump Compartment. Ensure minimum disturbance of the fluid lines, and that the stopper is replaced firmly afterwards.

The granules in the exhaust line scrubbing bottle for the vacuum pump should also be checked monthly and replaced every 3 to 6 months, according to how saturated they become. They will change colour and cake as they become saturated. There is another bottle next to the fume scrubbing granule bottle which acts as a moisture trap. This should be emptied if any condensates have accumulated in it.

YearlyThe HEPA filter should be replaced annually during preventative maintenance. However, the filter should also be replaced should an unusually strong acetic acid smell be detected when the system not in use. Switch the Isolator Switch to off. Remove reagent or waste vessels and pull the Filter Unit gently forward to within easy reach, and loosen the catches on the lid. Have a suitable sealable plastic bag to hand, and quickly remove the Filter Unit lid and bag the used cartridge. Insert the new cartridge, replace the lid and return the Filter Unit to its usual position. Switch the Isolator Switch to the on position and test to see if the filter fan is working as expected.

The moving parts of the Origa Towers are long-term lubricated. The manufacturer recommends a check and if necessary a change of wear parts, after an operation time of 12 months or 300km travel.

Other parts like soft tubing, peristaltic pump heads and the vacuum pump will be replaced preventively every other year as part of the maintenance service.



Emptying the Vacuum Bottle

The vacuum bottle cap has 3 tubes connected to it which will get twisted with the cap. It is therefore recommended to turn the bottle rather than the cap in order to open it. As there is vacuum in the bottle, you will meet resistance when trying to loosen the cap. For this matter we have installed a manual escape valve that allows air into the bottle. If possible, switch off the instrument when emptying the vacuum/waste bottle to avoid running the vacuum pump for an extended period unnecessarily.

The manual escape valve can be turned in either direction to let air in but there is a position which shorts the vacuum pump, fooling the vacuum gauge into thinking there is full vacuum in the bottle.

Image 1, opposite shows the position required for the instrument to operate properly.If you open the valve to the left (image 2), air will flow in but the pump air line will remain connected to the bottle (and the open air). In this position, it will be easy to detect that the valve has been accidentally left open.If you open the valve to the right (image 3), you will short the vacuum air line and the vacuum gauge will only measure the vacuum inside the tubing!

1. CORRECT OPERATING POSITION

2. RECOMMENDED EMPTYING POSITION

3. AVOID OPENING TO THIS POSITION



Appendix A: HMI Error Messages

Reacting to Alarms in GeneralIf for any reason an alarm sounds and an ERROR message appears on screen, then:

Press OK button once to silence the alarm.

Perform any corrective measures if possible (e.g. refill a reagent bottle or rearrange the buckets on the carousel)

(Press the Blue button and then the OK button if the main door was opened and allow the system to re-position itself)

When prompted, press OK to resume the cycle (allow the weight filter a few seconds to update if you have just replaced a bottle on the scales).

Note: This step is important so that the error gets cleared. You might have problems if you fail to press the OK button for the second time and go straight to Manual Control or Reset Cycle.

If necessary, at this point the cycle can be paused or reset (aborted) to start a new one.

It is generally safe to power off at any time but this should be left as a last resort, in particular if samples are in danger of getting compromised. Most small recovery/datum glitches can be overcome by opening and closing the door and subsequently pressing the blue and OK buttons as prescribed above. The support team at Genial are always a phone call away to help troubleshoot any problems and appreciate the urgency involved in certain stages of the harvest so don't hesitate to phone if you are having trouble operating the system.

Error MessagesERROR! PICK/PLACE SWING FAILURE ERROR! ASPIRATION SWING FAILUREERROR! HYPOTONIC SWING FAILUREERROR! FIXATIVE SWING FAILURE

This error normally only appears in Manual Mode when a tower is commanded to turn to a position which another one is already occupying. Other instances indicate that there has been a sensor failure or a drop of the position signal. Opening and closing the door, followed by pressing the Blue and OK buttons during a cycle forces the system to issue new signals by homing automatically and returning to the last position without having to go back to command a datum routine.

ERROR! ASPIRATION CLOG - CHECK PROBES!

The system has detected that the levels are not changing as expected in response to aspiration. Probes operate in pairs and if one probe clogs, the other automatically manages so processing can continue uninterrupted. If both become clogged, and the automatic cleaning routine fails to solve the problem, processing is halted and the alarm is raised. Before aborting the harvest, check that there aren’t any droplets bridging a pair of probes. Check the small blue box on the Aspiration head; it has a red LED for each pair of probes and it will light up while liquid is bridging them or they are immersed. Also check the level on the vacuum gauge which is located on the right wall of the reagent chamber. It should normally hover between -0.4 and -0.6 bar. If no LEDs are lit inside the blue box and the vacuum pressure is fine, try resuming the cycle in case the



problem has cleared. If the problem persists it will be necessary to abort the cycle and perform a manual wash operation. Recall the advice regarding Datuming the system at this point. If successive washing fails to clear the blockage, perform a visual inspection of the probes and report the problem to Genial.

ERROR! CHECK PICK/PLACE LIMIT SWITCHERROR! CHECK ASPIRATION LIMIT SWITCHERROR! CHECK HYPOTONIC LIMIT SWITCHERROR! CHECK FIXATIVE LIMIT SWITCH

This is usually a sign that the travel of the “arms” has gone as far as they are permitted to go, and have closed the contact of the limit switch. This prevents them from going further and damaging themselves or the gears of the driving motors. It is usually a sign they they've either started from the wrong place or one of the engineering or protocol parameters has been set beyond its limits. A Datuming operation is required here.

ERROR! VIBRATION - Check centrifuge

Usually the centrifuge is balanced, smooth and quiet. This alarm requires the cover to be retracted and a visual inspection made. The centrifuge cover can be manually opened as long as no probes are inside a bucket on the carousel. If the imbalance was caused by incorrect loading of the test tubes in the buckets, these can be checked and rectified but they must be returned to the same position they were occupying on the rotor. The centrifuge cover will close automatically once the main door is closed and the cycle is resumed. If the imbalance occurs halfway through a harvest it can be a symptom of another problem which went unnoticed, such as an aspiration failure or a pick&place error or one of the reagents failing to get pumped from the bottle to the test tubes.

P&P FAIL! - Put bkt in slot under grip

This can happen if a bucket does not appear where expected or if the grippers fail to pick up a bucket for any reason. Check the geometry of the load in the carousel and the rotor. If there is a bucket on the grippers, remove it by pulling up the release button above the gripper arms and place it in the slot under the grippers, be it over the carousel or the rotor. If there is a bucket already in that slot, check that it is sitting squarely in its location. Close the door and resume operation, observing that the pick&place sequence continues normally. Pick and place failures are mainly due to incorrect bucket positioning but can also be a symptom of the carousel or rotor not homing properly or of the grippers being knocked out of alignment.

WARNING! Check vacuum bottleWARNING! Check waste bottleWARNING! Check methanol bottleWARNING! Check acetic bottleWARNING! Check Hypotonic bottle

These warnings are directly related to the weigh scales and liquid levels in the bottles. An alarm will sound if the waste or vacuum bottles are filled beyond their threshold or if one of the reagent bottles is running too low. Refill/empty bottles as required and allow the weight filter a few seconds to settle before resuming operation.

ERROR! ASPIRATION PROBES CRASHEDERROR! HYPOTONIC PROBES CRASHEDERROR! FIXATIVE PROBES CRASHED

Each probe head has a sensor which will be triggered when the probes are pushed up if they encounter an object as they travel down. Check first that the probes haven’t suffered any damage, slippage or distortion and then see that the bucket being processed at that station doesn’t have jammed flaps and that its lid is correctly closed. If it looks safe to resume, do so checking the alignment of the probe head with the bucket as they come into contact with each other. A collision might also occur if incorrect parameters are set and the probes bottom out inside the wash station or the test tubes.



Appendix B: Technical Specif ica-tion

PowerCellsprint needs to be connected to mains supply electricity (240vAC/50 Hz or 110vAC/60Hz). It draws approximately 7-8 amps at this voltage so it is feasible to protect the unit with a small UPS to preserve a harvest in process during a temporary power outage. The rating of the UPS required would depend upon how long is the harvest being protected. Some establishments have their own emergency backup generators which come online after a short delay. and it might be that the UPS has only to cover processing during this interval.

Humidity & TemperatureThe range should be between 40% - 60% with no condensation. The maximum rate of change should be no more than 6%.

A suitable temperature range is 18°c - 24°c with a rate of change not exceeding 15°c per hour. If the temperature fluctuates in a repetitive manner, this figure should be reduced to 5°c per hour.

(Note: The above figures refer to the mechanical and electrical operation of CellSprint. No recommendation or expectation can be made or given regarding the effects of temperature and humidity upon the quality of harvests themselves.)

Weight400kg approx. when loaded

SizeHeight: 1870 mm (With door fully raised: 2370mm)Depth: 754mm (with doors open: 1215mm)Width: 1200mm (with pump chamber door opened to fullest extent: 1820mm)

Air FiltrationSingle cartridge containing:

Filter 1 (Coarse): 95% intercept for particles >= 3µm

Filter 2 (HEPA): 99.997% intercept for particles >= 0.3µm

Filter 3 (Activated charcoal). scrubbing of chemical fumes

Reagent BottlesCaps available for bottles with GL-45, GL-32 and 38-430 threads can be fitted according to local standard.

Test TubesMost conical and round bottom centrifuge tubes of 12 to 15 ml capacity are acceptable for use in CellSprint. Leighton tubes are also admitted but more careful setup is required.



Appendix C: EC Declaration of Conformity

Genial Genetic Solutions Ltd

EC Declaration of Conformity

This declaration of conformity is issued under the sole responsibility of the manufacturer.

Genial Genetic Solutions Ltd conforms to the relevant provisions of the EC Council Directive 93/42/EEC

Manufacturer:Genial Genetic Solutions Ltd

UK Registration: 4314936

Manufacturer’s Address: Genial Genetics P.O. Box 3751 Chester CH1 9UF UK

Device: CellSprint™ CSE-24 and CSP-48

Description: Automated suspension culture metaphase harvester

EC Product Class: Class I Medical Device (93/42/EEC)

Quality Management Representative: Mr C Bunn

Regulations and standards applied:

- EN 13949-1

CE marked, encompassing:

- Low Voltage Directive (LVD) 2006/95/EC- Waste Electrical and Electronic Equipment (WEEE) - 2002/96/EC- Electromagnetic Compatibility (EMC) – 89/336/EEC- Restriction of Hazardous Substances (RoHS) – 2002/95/EC- ISO 9001:2000 Certified by Lloyd’s Register QA for “The manufacture, supply and maintenance of automated sample processing equipment”

A full technical file is available for the CellSprint™ upon request.

Signature: N/A

Title: N/A



cellsprint user guide model cs24 and cs48 v4 - bionova | tec and c… · user guide genial genetic...

Documents