26530310 casy counter model dt-operators...

CASY Counter Model DT Operator‘s Guide

For life science research only.

Not for use in diagnostic procedures.

26530310 CASY Counter Model DT-Operators Guide.indd 126530310 CASY Counter Model DT-Operators Guide.indd 1 31.03.2010 13:38:1131.03.2010 13:38:11

3

Table of Contents

Prologue 5

I. Revision History .................................................................................................................................................5

II. Contact Addresses ...........................................................................................................................................5

III. Declaration of Conformity ............................................................................................................................6

IV. Warranty .................................................................................................................................................................6

V. Trademarks ...........................................................................................................................................................6

VI. Intended Use ........................................................................................................................................................6

VII. Preamble ................................................................................................................................................................7

VIII. Usage of the Instrument Operator‘s Guide .........................................................................................7

IX. Conventions Used in this Operator‘s Guide .......................................................................................8

X. Warnings and Precautions ...........................................................................................................................9

XI. Disposal of the Instrument ....................................................................................................................... 11

1. Introduction 13

1.1 The measuring principle of the CASY Model DT. ......................................................................... 13

1.2 CASY Model DT ............................................................................................................................................... 14

2. Device confi guration 15

2.1 Measuring unit ................................................................................................................................................ 15

2.2 Measuring capillary and external electrode ................................................................................... 17

2.3 Pressure system and power supply ..................................................................................................... 17

2.4 Control panel .................................................................................................................................................... 18

3. Start-up 19

3.1 Choosing the location ................................................................................................................................. 19

3.2 Cabling ................................................................................................................................................................. 19

3.3 Switch-on ........................................................................................................................................................... 20

3.4 Preparing for measurement ..................................................................................................................... 21

4. Caring for your CASY Model DT 22

4.1 Weekly cleaning routine ............................................................................................................................. 22

4.2 Cleaning the cylinders and the sampling chamber .................................................................... 23

4.3 How to set CASY Model DT out of operation .................................................................................. 23


4CASY Counter Model DT

Table of Contents

5. Operation of CASY Model DT 24

5.1 General ................................................................................................................................................................. 24

5.2 Menu Control - MAIN MENU .................................................................................................................. 24

5.3 MAIN MENU - Select Setup .................................................................................................................... 25

5.4 MAIN MENU - Edit Setup .......................................................................................................................... 26

5.5 MAIN MENU - Select Capillary .............................................................................................................30

5.6 MAIN MENU - Set Date and Time .......................................................................................................31

5.7 MAIN MENU - System Settings ............................................................................................................ 32

5.8 MAIN MENU - Service Menu ................................................................................................................. 33

5.9 Cursor control using the control panel keys ..................................................................................34

6. Performing and evaluating measurements 35

6.1 Preparing samples ......................................................................................................................................... 35

6.2 Measuring samples and displaying the measuring results ...................................................36

6.3 Points to be observed when performing measurements .........................................................38

6.4 Setting the cursor positions ..................................................................................................................... 43

6.5 Outputting the results ..................................................................................................................................46

7. Appendix 47

7.1 Error messages ................................................................................................................................................ 47

7.2 Data output via the serial interface .....................................................................................................48

7.3 Technical data on CASY Model DT .......................................................................................................49


5Prologue

Prologue

I. Revision History

Version Revision Date

1.0 December 2009

1.1 (editorial changes) March 2010

© Copyright 2010, Roche Diagnostics GmbH. All rights reserved. Information in this document is subject to change without notice. No part of this document may be reproduced or transmitted in any form or by any means, electronic or mechanical, for any purpose, without the express written permission of Roche Diagnostics GmbH.

Questions or comments regarding the contents of this Operator’s Guide can be directed to the address below or to your Roche representative.

Roche Diagnostics GmbHRoche Applied ScienceCustomer SupportNonnenwald 282377 Penzberg, Germany

Every effort has been made to ensure that all the information contained in the CASY Counter Model DT Instrument Operator’s Guide is correct at the time of printing.

However, Roche Diagnostics GmbH reserve the right to make any changes necessary without notice as part of ongoing product development.

II. Contact Addresses

Manufacturer Roche Innovatis AGKrämerstraße 22D-72764 ReutlingenGermany

Distribution Roche Diagnostics GmbHSandhofer Straße 116D-68305 MannheimGermany

Distribution in USA Roche Diagnostics 9115 Hague Road PO Box 50457 Indianapolis, IN 46250 USA

Revision History



III. Declaration of Conformity

According to Council directive 73/23/EWG and 89/336/EWG we declare, that the CASY Model DT is conform with standards and regulations as listed below:

EN 61010-1/03.94

IEC 61010-1/09.90 and Amendment 1/09.92

(according to Classifi cation VDE 0411, part 1/09.94)

IV. WarrantyInformation on warranty conditions are specifi ed in the sales contract. Contact your Roche representative for further information.

Any unauthorized modifi cation of the instrument invalidates the guarantee and service contract.

V. Trademarks454, 454 LIFE SCIENCES, 454 SEQUENCING, CASY and INNOVATIS are trademarks of Roche.

Other brands or product names are trademarks of their respective holders.

VI. Intended UseThe CASY Counter Model DT is intended for highly standardized and accurate counting of beads used by the 454 Sequencing System. The technology of the CASY Model DT combines a proven particle measurement technique, referred to as the resistance measurement principle, with a modern method of signal evaluation, pulse area analysis.

The CASY Model DT Instrument is intended for life science research and must be used exclusively by laboratory professionals who are trained in laboratory techniques and have studied the instructions for use of this instrument. The CASY Model DT Instrument is not intended for use in diagnostic procedures.

Declaration of Conformity


7Prologue

VII. PreambleBefore setting up operation of the CASY Model DT, it is important to read this Operator’s Guide thoroughly and completely. Non-observance of the instructions contained herein or performing an operation not stated in this Operator’s Guide could involve safety hazards.

VIII. Usage of the Instrument Operator’s GuideThis Operator’s Guide describes the operation of the CASY Model DT Instrument and contains the following chapters:

Chapter A Introduction contains a short introduction to the measurement principle of the technology of the CASY System.

Chapter B Device confi guration contains the description of the CASY Model DT instrument’s components.

Chapter C Start-up describes the installation of the CASY Model DT and instructions for Start-up and preparation for measurements.

Chapter D Caring of your CASY Model DT describes the maintenance procedures that are required for the CASY Model DT Instrument.

Chapter E Operation of CASY Model DT describes the general operation of the CASY Model DT instrument including the explanation of the parameters that can be modifi ed.

Chapter F Performing and evaluating measurements contains instructions for sample preparation and describes the procedure for performing a measurement. Furthermore the chapter describes the data output.

Chapter G Appendix contains trouble shooting and information in regards to data output via serial interface and technical information.

Preamble



IX. Conventions Used in this Operator’s Guide

Text Conventions

To impart information that is consistent and memorable, the following text conventions are used in this Operator’s Guide:

Numbered Listing Describes the steps in a procedure that must be performed in the order listed.

Italic type Important notes and information notes are shown in italic type.

Symbols

In this Operator’s Guide symbols are used as a visual signal to highlight important points.

Symbol Heading Description

WARNING This symbol is used to indicate that noncompliance with instructions or procedures could lead to physical injury or even death or could cause damage to the instrument.

BIOHAZARD This symbol indicates that certain precautions must be taken when working with potentially infectious sample material.

IMPORTANT NOTE Information critical to the success of the procedure or use of the product.

INFORMATION NOTE Additional information about the current topic or procedure.

�� Table continued on next page.

■ End of table.

The following symbols appear on the instrument:

Symbol Heading Description

WEEE Electrical and electronic equipment marked with this symbol are covered by the European Directive WEEE.

The symbol denotes that the equipment must not be disposed off in the municipal waste system.

Conventions Used in this Operator‘s Guide


9Prologue

X. Warnings and Precautions

Handling Requirements

It is important that the following safety instructions and guidelines be observed in order to ensure safe and reliable operation of CASY Model DT:

� Place the instrument on a solid, dry work table that is exposed to neither strong mechanical vibrations nor high-volume noises (e.g. ultrasonic baths).

� Choose the installation site so that CASY Model DT is protected from mechanical damage and water and solvent spillage.

� Choose a room that is as smoke and dust-free as possible and that has a constant room temperature of between 15°C and 32°C.

� Do not place CASY Model DT in the vicinity of devices that generate strong electrostatic or electromagnetic fi elds. This includes specifi cally computer monitors that work with high-voltage picture tubes, brush motors, fl ickering fl uorescent lamps, arcing contacts, water baths, gas chromatographs etc. In the event of any alternative site being available, maintain the greatest possible distance from such devices.

� Only trained personnel in a controlled laboratory environment should operate the CASY System.

� All services and repairs should be performed only by authorized service technicians.

� Always ensure that there is suffi cient CASY ton in the CASY ton storage container.

� Clean the containers before reusing them.

� Always observe the safety regulations when handling CASY ton and CASY clean.

� Waste contaminated with tissue or cells, e.g. waste container, CASY cups, pipette tips, etc., must be disposed of according to the recommended rules for handling biohazard materials.

� Any leaks in the Liquid Management system must be repaired immediately. Contact your distributor or authorized service technician.

� Always ensure when the CASY Model DT fl uid system contains a saline solution that it does not dry out to prevent malfunction or damage to the fl uid system and valves caused by salt crystals.

� Only use CASY clean for cleaning the CASY Model DT fl uid system.

� Always wear protective clothing, particularly gloves, when handling samples.

� Dirt or dust particles should not gain access into the device.

� Ensure that all equipment required for sample preparation, e.g. CASY cups, pipette tips, etc., are stored in a dust-free environment.

� Filter the CASY ton through a sterile fi lter with a pore size ≤ 1.2 µm and use a new CASY cup for every measurement.

For your own safety, please consider all biological material as potentially infectious. Handling and disposal of such material should be performed according to local safety guidelines. Spills should be immediately disinfected with an appropriate disinfectant solution to avoid spreading infection to, and contamination of, laboratory personnel or equipment. When working with potentially infectious material, always use protective gloves (powder-free).

� Please refer to the section on Caring for your CASY Model DT for instructions on cleaning the CASY Model DT Instrument.

Warnings and Precautions



General Precautions

Make sure that the main switch can be reached easily. Inappropriate location of the instrument can cause incorrect results and damage to the equipment parts. Follow the installation instructions in the chapter Start-up carefully. Keep all potentially infl ammable or explosive material (for example, anesthetic gas) away from the instrument. Spraying liquid on electrical parts can cause a short circuit and result in a fi re.

During fi refi ghting operations, disconnect the power line of the CASY Model DT Instrument.

Electrical Safety

The CASY Model DT Instrument is designed in accordance with Protection Class I (IEC). The chassis/housing of the instrument is grounded to protection earth (PE) by means of a cord. For protection against electrical shock hazards, the instrument must be directly connected to an approved power source such as a three-wire grounded receptacle for the 110 V or 230 V line. Do not use an ungrounded receptacle; have a qualifi ed electrician replace the ungrounded receptacle with a properly (PE) grounded receptacle in accordance with the local electrical code. Do not use an extension. Any break in the electrical ground path, whether inside or outside the instrument, could be hazardous. Under no circumstances should the user attempt to modify or deliberately defeat the safety features of this instrument. If the power cord becomes cracked, frayed, broken, or otherwise damaged, replace it immediately with the equivalent part from Roche Diagnostics.

Warnings and Precautions


11Prologue

XI. Disposal of the InstrumentAll electrical and electronic products should be disposed of separately from the municipal waste system. Proper disposal of your old appliance prevents potential negative conse-quences for the environment and human health.

The instrument must be treated as biologically contaminated hazardous waste. Final disposal must be accomplished in a way that does not endanger waste handlers. As a rule, such equipment must be sterile before it is passed on for fi nal disposal.

For more information contact your local Roche Support personnel.

The instrument should also be decontaminated prior to shipping it for outside service or repairs.

Components of your CASY Model DT which are marked with the crossed-out wheeled bin symbol are covered by the European Directive 2002/96/EC (WEEE).

These items must be disposed of via designated collection facilities appointed by government or local authorities.

For more information about disposal of your old product, please contact your city offi ce, waste disposal service or your local Roche Support personnel.

It is left to the discretion of the responsible laboratory organization to determine whether the CASY Model DT measuring unit and the control panel are contaminated or not. If contaminated, treat the control panel in the same way as described for the cylinders and the sampling chamber.

Disposal of the Instrument


13Introduction

AAIntroduction1.

CASY Model DT is a German development based on extensive experience in analytical working methods in the fi elds of cytobiology, microbiology, biochemistry and physics. Its ergonomic and easy operation were developed specifi cally for day-to-day, routine use. Standardized counting takes place simultaneously in three size ranges. This ensures highly standardized and accurate counting of beads used by the 454 Sequencing System.

The measuring principle of the CASY 1.1 Model DT.

The technology of the CASY Model DT combines a proven particle measurement technique, referred to as the electrical sensing zone technique (ISO 13319), with a modern method of signal evaluation, Pulse area analysis.

Pulse area analysis was developed in 1987 by Schärfe System on the basis of Digital Pulse Processing (DPP), a process employed for the digital recording of electrical signals for more than 40 years. For particle analysis, DPP is combined with a high scanning rate and a highly-standardized method of analysing particle signals. This combination facilitates unprecedented measuring range dynamics and guarantees maximum resolution and excellent reproducibility for all measuring parameters.

Measurement is performed by suspending the particles in CASY ton, an electrolyte developed specifi cally for particle counting and aspirating them through a precision measuring pore of defi ned geometry at a constant fl ow speed. The precision measuring pore is designed as a hole within an artifi cial precious stone, which is cast inside the capillary body. During the measurement process, a pulsed low voltage fi eld with 1 MHz is applied to the measuring pore via two platinum electrodes. The electrolyte-fi lled measuring pore represents a defi ned electrical resistance. During their passage through the measuring pore, the particles displace a quantity of electrolyte corresponding to their matrix volume. Particles can generally be considered isolators, an increased level of resistance is achieved over the measuring pore. This resistance is a dimension for the volume of the particles. It is important that the particles are passing through the measuring pore individually.

The measuring signal is scanned by the CASY Counter at high frequency. As well as the amplitude of the measuring signal, the CASY Counter also records the shape of the signal as a whole. The CASY Counter calculates the integral of the measuring signal from the individual measurements. The calculated signal areas are evaluated by a standardized method and accumulated in a multi-channel analyser (pulse area analysis).

The signal amplifi er technology, developed specifi cally for CASY Model DT , guarantees excellent measuring range dynamics (> 1 : 70000 in terms of volume and > 1 : 40 in terms of diameter respectively). Measuring range dynamics is described as the ratio from the smallest to the largest particle that can be analysed simultaneously. This guarantees that the entire physical measuring range of the used measuring capillaries from approx. 2% to approx. 80% of the measuring pore diameter is always in view, from very small particles to very large particles.

The measuring principle of the CASY Model DT.



AA CASY Model DT1.2 CASY Model DT combines a high degree of ergonomics with functionality. At the push of a button, the electronics control the measuring stand, record the measuring data, evaluate the measuring data via a multi-channel analyser, calculate and display the measuring results on the control panel. All measuring parameters can be set through a minimum of stages. Up to 20 different settings can be stored and remain available the next time the device is switched on.

To output the data, a HP-compatible printer (PCL) can be connected directly to the device. Furthermore, the data for documentation and printing can be transferred via the serial interface to a connected computer.

CASY Model DT


15Device confi guration

BB

Device confi guration2.

CASY Model DT comprises the following functional units:

� Measuring unit consisting of measuring unit, pressure systems and mains power supply (see Figure 1).

� Measuring capillary (see Figure 1).

� Control panel with large display and 13 control keys (see Figure 2).

Measuring unit, pressure system and mains power supply are accommodated in two separate metal cylinders. This guarantees maximum protection for the highly-sensitive measuring electronics against interference and enables the measuring capillaries to be arranged in one large, open sampling chamber.

Measuring unit2.1 The measuring unit (see Figure 1) contains the measuring electronics, the fl uid system and the valves required to control the measuring process. The close monitoring of the measuring volume (200 µl or 400 µl) required to determine the concentration is carried out by a light-barrier monitored, calibrated precision vertical tube. CASY Model DT handles larger measuring volumes (maximum 4 ml) by employing a multiple measurement system. The results of the individual measurements are accumulated. CASY Model DT operates almost dead-volume free, i.e., the required sample quantity is only slightly larger than the measuring volume. The control and monitoring of the measuring process are designed to exhibit maximum protection against interference. Any faults that do occur are automatically detected by CASY Model DT and notifi ed to the user.

The sampling chamber accommodates the following elements:

� Measuring capillary

� External electrode

� Sample platform

� Waste container (left)

� Storage container (right)

Measuring unit



BB

Pressure System and Power Supply

Light-Emitting Diodes

Waste Container

Base

Precision Measuring Capillary

Calibrated Vertical Tube

Measuring Unit

External Electrode

Storage Container

Sample Platform

Figure 1: Schematic of CASY Model DT, measuring stand, frontal view

The measuring unit is controlled by the control panel (see 3.4 and Figure 2). An inspection window, which also enables the vertical tube to be monitored visually, is situated above the sampling chamber. The inspection window accommodates three light-emitting diodes, which indicate the fi lling level of the vertical tube. The green light-emitting diode indicates the start of a measurement, the lower red light-emitting diode indicates when the 200 µl level is reached and the upper red light-emitting diode indicates when the 400 µl level has been reached.

Before the start of each measurement, the contents of the measuring capillary are emptied into the waste container. The sample volume is then aspirated through the measuring capillary under constant low pressure. The progress of the meniscus can be tracked in the inspection window. The time required to reach the light barriers depends on the diameter of the used measuring capillary and is precisely monitored by CASY Model DT. This process enables potential defects resulting from measuring capillary blockages to be automatically detected. During the measurement process, the vertical tube is also monitored for disturbances caused by air bubbles.

Blockages can be removed by running a clean cycle, which, in addition to refi lling the internal fl uid system, also actively counter-fl ushes the measuring pore (see 4.4). The required CASY ton is sucked in from the storage container and then emptied into the waste container. The entire volume of the internal fl uid system is approx. 2 ml.

Measuring unit


17Device confi guration

BB

Measuring capillary and external 2.2 electrode

The external electrode (see Figure 1) is from platinum, arranged before the measuring capillary, can rotate freely and be pulled downwards if necessary.

The measuring capillary (see Figure 1) is from high-resistance plastic. It is secured to the sampling chamber cover by a cap nut. At the lower end of the capillary body, an artifi cial precious stone is cast with the precision measuring pore. The measuring pore has a cylindrical geometry with a diameter to length ratio of 1:1. The axial arrangement of the hole minimises the risk of blockage and prevents impurities from accumulating inside the capillary body.

To remove the measuring capillary, fi rst carefully rotate the external electrode forwards. Then release the cap nut and remove it with the measuring capillary by pulling downwards. In order to prevent the measuring capillary becoming contaminated with grease and dust, try to avoid touching its lower end, which is immersed in the sample.

To re-assemble, screw in the cap nut manually (do not use a tool)! Ensure that the tube extending inside the measuring capillary is not buckled or pinched during assembly. Then, carefully rotate the external electrode, without bending, returning it to its original position. Ensure that the background is monitored (see 4.4) before commencing the measurement.

Unused measuring capillaries must always be cleaned thoroughly before being stored. Dried residues in the measuring pore can result in subsequent blockages. This can be prevented by placing the measuring capillaries in CASY clean and leaving overnight. The following day, rinse the measuring capillaries carefully using CASY clean and then fl ush repeatedly with distilled water. Blow the fl uid from the measuring capillaries and store in a clean place.

The measuring capillaries should be cleaned using only the CASY clean agent specifi ed by Roche Innovatis AG. Other agents may destroy the measuring capillaries. Roche Innovatis AG is not liable for any damage caused by using alternative cleaning agents.

Pressure system and power supply2.3 The pressure system is accommodated together with the power supply in its own cylinder situated behind the measuring unit (see Figure 1). A no-maintenance vacuum pump, combined with the latest pressure sensors, guarantee that the vacuum required to aspirate the sample remains constant, irrespective of any external pressure. The pressure system also interacts with the valves on the measuring unit to control all fl uid movements required to empty the vertical tube, replenish the fl uid system and counter-fl ush the measuring capillary.

The power supply for the CASY Model DT measuring electronics contains special fi lters, which ensure that the mains power supply is afforded maximum interference protection.

Measuring capillary and external electrode



BB

Control panel2.4 The control panel (see Figure 2) consists of a display and a membrane keyboard exhibiting 13 keys. In measuring mode, the display indicates the measuring results. All measuring and output parameters can be displayed and adjusted through the menu control.

The keyboard is divided into three functional areas. The top right-hand area accommodates the keys used to enter the measuring and output parameters. The bottom right-hand area accommodates the keys used to control the measuring stand and output the results to the printer. On the left are the keys used to position the evaluation cursors. All keys have a clearly defi ned pressure point.

Display

Defi nition of key functions:

MENU: Starts the menu control.

CANCEL: Cancels a measurement or entry.

ENTER: Confi rms an entry.

NEXT: Continue to next number.

� : Increases number / change selection.

� : Increases number / change selection.

PRINT: Output to printer and/or serial interface.

CLEAN: Starts one or several cleaning cycles.

START: Starts a measurement.

L-CURSOR: Positions left cursor.

: Shifts cursor to the right.

: Shifts cursor to the left.

R-CURSOR: Positions right cursor.

Figure 2: CASY Model DT, Control panel

For adjusting the contrast of the CASY Control Panel display, you will fi nd an adjustment wheel located on the right side of the CASY Control Panel.

Control panel


19Start-up

CC

Start-up3.

Choosing the location3.1 CASY Model DT is a precision measuring instrument. To ensure fault-free measuring operation, you must choose the location according to the following criteria:

� Place the instrument on a solid, dry work table that is exposed to neither strong mechanical vibrations nor high-volume noises (e.g., ultrasonic baths).

� Choose the installation site so that CASY Model DT is protected from mechanical damage and water and solvent spillage.

� Choose a room that is as smoke and dust-free as possible and that has a constant room temperature of between 15°C and 32°C

� Do not place CASY Model DT in the vicinity of devices that generate strong electrostatic or electromagnetic fi elds. This includes specifi cally computer monitors that work with high-voltage picture tubes, brush motors, fl ickering fl uorescent lamps, arcing contacts, water baths, gas chromatographs etc. In the event of any alternative site being available, maintain the greatest possible distance from such devices.

Cabling3.2 Connect your CASY Model DT to an interference-free measuring instrument socket. Under no circumstances should you operate CASY Model DT from the same socket as laboratory equipment that works with high switching loads (autoclaves, centrifuges, agitators etc.). You must also ensure that all laboratory equipment situated in the same vicinity is interference-free/fault-free. The following connection cables and/or control panels must be connected.

� Data line for control panel (9-pin sub-D plug).

� Mains power cable.

� Printer, if available (25-pin sub-D plug).

All connections are clearly labeled to prevent confusion.

Before switching on for the fi rst time, ensure that the mains voltage set on the voltage selector on the rear of the device is consistent with your mains power supply. If it is necessary to convert the mains power supply, please observe the information on the primary fuses (see 8.3). Roche Innovatis AG accepts no liability for damage caused by operating the device from an inappropriate power supply.

Choosing the location



CC

Switch-on3.3 Fitted to the rear panel of CASY Model DT is a green power switch, which when turned to ON, illuminates and indicates that the device is live. After being switched on, CASY Model DT automatically performs all the required initialisation procedures. Specifi cally, the CASY Counter is subjected to a complete self-test during which the hardware, pressure system and all memory modules (program, calibration, setups) are checked. The following screen appears when the CASY Counter is powered up:

SCHÄRFE SYSTEM

CASY DTCell Counter

COMPLETE SYSTEM SELFTEST

Hardware : ---> ok

Pressure System: ---> ok

Memory : running

When the initialisation procedure is complete, your CASY Model DT will display the standard screen for the measuring results. All values are initially set to zero. The previously used setup is loaded automatically.

Before switching on your CASY Model DT, ensure that the control panel is connected and properly bolted to the measuring stand. Removing the plug from the control panel when the measuring stand is switched to ON may result in damage to the display. Roche Innovatis AG accepts no liability for damage caused by severing a live connection between the measuring stand and the control panel.

Switch-on


21Start-up

CC

Preparing for measurement

Preparing for measurement3.4 CASY Model DT is factory-calibrated. Preparing for measurement is therefore limited to the following steps:

� Replenishing the storage container with clean CASY ton.

� Emptying the waste container.

� Fitting (see 3.2) the required measuring capillary where necessary.

� Selecting an appropriate setup (see 6.3) or manually inputting the parameters (see 6.4)

� Monitoring the background.

You must monitor the background of your CASY Model DT at regular intervals. Problems resulting from choosing an unsuitable location and also those relating to impurities in the CASY Counter manifest themselves in too high a background.

Background monitoring

1 Place a CASY cup containing CASY ton below the measuring capillary.

2 Press and hold the CLEAN key down until the clean counter indicates 3. Releasing the key triggers three clean cycles.

3 Place a CASY cup containing CASY ton below the measuring capillary.

4 Press the START key to initiate a measurement.

■

To guarantee that the CASY cup is fi xed, you can rotate the sample platform one turn so that the recess is not longer pointing forwards. This secures the cup. If you perform the background monitoring with a setup different from the setup stored at delivery of the system (setup no. 00), please ensure that the dilution is set to 1 and that the counts in the entire range of the X-axis are monitored.

Using the 150 µm measuring capillary, the background should not exceed 100 Counts/ml (typically 10 - 50). Too high a background may result from impurities in your CASY ton or not having used a new CASY cup. Filter the CASY ton through a sterile fi lter with a pore size of ≤ 1.2 µm and use a clean CASY cup for every measurement. Repeat steps 1 to 4 with freshly fi ltered CASY ton. If the CASY Counter fails to become clean even after several attempts, you should carry out the Weekly Cleaning routine (see 5.1). If this exercise fails to produce the desired result, please check that the location of your CASY Model DT satisfi es the criteria indicated in 4.1.

You must ensure that all the vessels used to prepare the samples, the CASY ton used to rinse the CASY Counter and dilute the samples and also any CASY cups used are free of grease and particle impurities. For routine preparation of the measuring samples, use a dispenser (10 ml) with integral fi lter (≤ 1.2 µm) wherever possible.

To avoid incorrect measurements, CASY cups should be used once only.



DD

Weekly cleaning routine

Caring for your CASY Model DT4.

Your CASY Model DT is designed to be low-maintenance; the fl uid system however requires regular attention to guarantee fault-free work over several years. Residues in the fl uid system may lead to blockages in the measuring pore, too high a background or other faults during operation. To avoid such errors, you must follow the cleaning procedure described below at least once per week. The recommended agent, CASY clean, is available from Roche Innovatis AG.

The CASY Counter should be cleaned using only the CASY clean agent specifi ed by Roche Innovatis AG. Never use aggressive cleaning fl uids on CASY Model DT. Roche Innovatis AG accepts no liability for damage caused by using other cleaning agents!

Weekly cleaning routine4.1

1 Fill two CASY cups with CASY clean.

2 Place one CASY cup under the measuring capillary.

3 Replace the storage container holding CASY ton (right-hand cylinder) with the second CASY cup.

4 Run three cleaning cycles (hold the CLEAN key down and select 3 cycles).

5 Perform a triple measurement using a measuring volume of 400 µl (START).

6 Leave your CASY Counter for at least 3 hours in CASY clean.

7 Replace the cleaning solution with two CASY cups containing distilled water.

8 Run three cleaning cycles (hold the CLEAN key down and select 3 cycles).

9 Perform a triple measurement using a measuring volume of 400 µl (START).

10 Repeat steps 7 to 9 to remove the cleaning solution completely.

11 Repeat steps 7 to 9 using CASY ton.

12 Your CASY Model DT is now operational again.

■

Duty of care:

Never leave samples under the measuring capillary! On completion of a series of measurements, place a CASY cup containing fresh CASY ton under the measuring capillary and run at least 3 clean cycles (hold the CLEAN key down and select 3 cycles).

Before switching off, always monitor the device’s background (see 4.4). Run a measurement using fresh CASY ton.


23Caring for your CASY Model DT

DD

Cleaning the cylinders and the sampling chamber

Cleaning the cylinders and the sampling 4.2 chamber

Clean the surfaces of your CASY Model DT regularly using a damp micro-fi bre cloth. It is important to remove salt solution spillages immediately. To do this, raise the sample platform and remove by pulling forwards. Methylated spirit can be used to remove organic impurities. Under no circumstances, however, may other organic solvents be used.

How to set CASY Model DT4.3 out of operation

If your CASY Model DT has not to be operated for several weeks, the fl uid system should be drained. Before shutting down, the fl uid system must be cleaned (see 5.1, Weekly Cleaning Routine). Follow this procedure to shut-down the CASY Counter:

1 Carry out steps 1 to 10 of the Weekly cleaning routine.

2 Replace the two CASY cups containing distilled water with two empty CASY cups.

3 Select the Service Menu option from the main menu.

4 Execute the Dry Liquid System command from the Service Menu.

5 Empty the waste container and the two CASY cups.

6 You can now switch off your CASY Counter.

7 Ensure that the CASY Counter is adequately protected from dust whilst it is shut down.

8 You should begin the Start-up procedure with the Weekly cleaning routine.

■

You must ensure that when the CASY Model DT fl uid system contains a saline solution, it is not allowed to dry out. This will prevent malfunction or damage to the fl uid system and valves caused by salt crystals.



EE

General

Operation of CASY Model DT5.

General5.1 CASY Model DT is operated using 13 control keys. Operation is divided into three distinct areas, each of which refl ects the spatial arrangement of the keys (see Figure 2.).

� Entering parametersUpper right-hand keys: MENU, CANCEL, ENTER, NEXT, �, �

� Measurement operation and output of resultsLower right-hand keys: START, PRINT, CLEAN.

� Evaluation with various cursor positions Left-hand keys: L-CURSOR, , , R-CURSOR.

Because up to 20 device settings can be permanently stored, parameter setting in most cases can be done by selecting an appropriate setup. Each time CASY Model DT is switched on, the previously used setup is loaded. All other setups are readily available through the menu function. In addition, the measuring and output parameters can be modifi ed to match the actual measurement and do not have to be stored in one of the setups.

Menu Control - MAIN MENU5.2 All CASY Model DT settings are selected with the MENU key. Pressing MENU displays the MAIN MENU.

MAIN MENU DT-2AB-9999 15.04.2002 10:15

* Select Setup

Edit Setup

Select Capillary

Set Date and Time

System Settings

Service Menu

USE MENU TO SELECT AND PRESS ENTER TO OPEN.USE CANCEL TO LEAVE SCREEN.

All menu control screens have the same structure. The name of the screen in upper case letters is contained in the left part of the header, which is separated by a horizontal line: MAIN MENU. Displayed to its right are the serial number of your CASY Model DT, the date and time. The lower border, also separated by a horizontal line, contains instructions on how the screen and individual keys operate.

Use the MENU key to select individual menu items. Pressing ENTER opens a menu item. Pressing CANCEL closes the MAIN MENU. If you overshoot the item you wish to select, you can move backwards by pressing and holding down the MENU key.

Irrespective of which menu control screen you are in, pressing CANCEL repeatedly returns you to the MAIN MENU and then to the result display.


25Operation of CASY Model DT

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MAIN MENU - Select Setup

MAIN MENU - Select Setup5.3 The Select Setup option opens the SETUP SELECTION screen. This screen enables you to select one of 20 permanently stored setups.

SETUP SELECTION DT-2AB-9999 15.04.2002 10:15

Capillary: 150 μm

Setup No: 00 01 02 03 04 05 06 07 08 09

*

10 11 12 13 14 15 16 17 18 19

USE NEXT TO SELECT SETUP AND PRESS ENTER TO LOAD.USE CANCEL TO LEAVE SCREEN.

The setups are numbered from 00 to 19. Each setup stores a complete set of parameters. The setups already in use to store parameters are tagged with *. If no setup is logged, the CASY Counter works with the factory-default set of parameters.

Pressing the NEXT key selects a setup. You may select only those setups that have been logged and tagged with *. Pressing and holding down the NEXT key enables you to move the selector mark backwards. Pressing ENTER displays the selected setup.

Further details on the setup screen can be found under Edit Setup (see 6.4). Check whether the selected setup matches your needs. Besides the measuring and evaluation parameters, each setup has a distinct name which you can defi ne and which is displayed in the top right-hand corner of the setup screen.

Press ENTER again to load the setup displayed. If you do not wish to load the setup displayed, you can return to Setup Selection by pressing CANCEL.

The setup parameters cannot be changed at this point. To edit a loaded setup, use the Edit Setup menu item described below (see 6.4).



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MAIN MENU - Edit Setup

MAIN MENU - Edit Setup5.4 Edit Setup opens the setup screen. All the parameters provided by CASY Model DT to defi ne the measuring conditions, calculate and output the measuring results, can be edited in this screen. The active parameters are always displayed. Any setup you wish to edit must fi rst be loaded through Select Setup (see 6.3).

SETUP NUMBER: 01 NAME: Beads

Capillary : 150 μmSample Volume : 3 x 400 μl

Dilution : 3.333e+03

Left Cursor : 15.00 μmRight Cursor : 25.00 μm

Display Mode : StandardTotal Counts/ml : Off

Interface : Parallel Print-Mode : Manual/PF On

USE MENU AND NEXT TO STEP. USE +/- TO EDIT.USE CANCEL TO LEAVE SCREEN OR ENTER TO CONFIRM CHANGES.

The setup screen is divided into three areas. The header, separated by a horizontal line, contains the SETUP NUMBER (00-19) and the NAME (setup name) you have defi ned. If the active parameters do not correspond to any of the stored setups, two forward slashes // are shown in place of the setup name and NOT SAVED in place of the setup name.

The block below, separated by another horizontal line, contains the measuring parameters. These parameters must be defi ned before a measurement takes place. They may not be retrospectively changed.

The main block below contains all evaluation parameters and also the output parameters, which defi ne the settings for outputting via a directly connected printer or via the serial interface. The parameters can be modifi ed following a measurement.

When a setup screen is opened, the input marker is located initially on the Dilution input fi eld. The current input position is indicated by a fl ashing, horizontal line below the characters which can currently be changed. From this point, you can use the MENU key to move forwards or backwards between the input fi elds. To move forwards, press the MENU key briefl y. The input maker skips forward to the next available input position. To move backwards, keep the MENU key pressed until the input marker reaches the position you require. Both methods move you to the start or the end of the screen when the last or the fi rst parameter respectively is reached.

When inputting numbers, you can move forwards from number to number using the NEXT key. To increase or decrease a number selected using NEXT, use the � and � keys. For all other parameters, the � and � keys can be used to select various pre-defi ned values. This also applies for the individual characters of the setup name.



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NAME (Setup name)

Each setup can be assigned a specifi ed name. Each name can be up to 10 characters long (letters and numbers). The setup name is output with the numerical measuring results both on the display and on the printout.

Measuring parameters

The measuring parameters must be set before the measurement takes place. They cannot be modifi ed once the measurement has been carried out. To change the measuring parameters, the sample must always be subjected to a second measurement!

Capillary: The capillary belonging to the processed setup is displayed here for information only. You cannot change its value. The system is specially used for bead counting for 454 Sequencing Systems therefore it is equipped with the 150 µm capillary which covers a size range from approx. 3,3 to 100 µm.

Sample Vol: Enter here the number of repeat measurements (1-10) and the measuring volume (200 µl or 400 µl). The individual measurements are automatically accumulated. The maximum measuring volume is 4.0 ml (10 × 400 µl). The set measuring cycles and the measuring volume are automatically considered as the particle concentration is calculated. You should in principle use the 400 µl setting.

Evaluation parameters

Dilution: The dilution factor is used to calculate the particle concentration in the original sample. It is entered in exponential format (e.g., for dilution 200, enter: 2.000e+02). Pressing NEXT enables the input marker to move from number to number. Holding this key down causes the input marker to move backwards.

Left Cursor: Left and right cursors separate the measuring range into three size categories, which are measured simultaneously. Depending on the Display Mode parameter (see below), percentage evaluations of the three size ranges can be displayed. The + and – keys can be used to position the evaluation cursor. Pressing NEXT enables the input marker to move from number to number. To change cursor position, you can also use the arrow keys on the control panel (see 6.9).

Right cursor: Left and right cursors separate the measuring range into three size categories, which are measured simultaneously. Depending on the Display Mode parameter (see below), percentage evaluations of the size ranges can be displayed. The + and – keys can be used to position the evaluation cursor. Pressing NEXT enables the input marker to move from number to number. To change cursor position, you can also use the arrow keys on the control panel (see 6.9).

Display Mode: In addition to the Standard setting (percentage calculation disabled), two percentage calculation settings (%Calculation, %Via) are available.

%Calculation: This setting calculates the percentage counts in the size range between left and right cursor in relation to the size range of between left cursor and the right-hand end of the measuring range, which is set to 100%. The percentage counts to the right of the right cursor are displayed accordingly. The two percentages must total 100%.

%Via: This setting will never be setup for counting beads used by the 454 Sequencing System. Therefore detailed description of %Via can be omitted.

Total/ml: This parameter can be either On or Off. In the On setting, besides the concentrations in the three individual count ranges, the concentration in the size range between the left cursor and the right-hand end of the measuring range is output.



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Output parameters

Interface: You can specify through the interface, whether after pressing PRINT, the data should be output through the parallel output – Parallel, through the serial output – Serial or through both simultaneously – P + S. The P + S setting enables you to print the data, whilst simultaneously transferring it to a connected computer. The connected printer must be PCL capable. Hewlett-Packard printers and all compatible products satisfy this requirement. More precise details on the serial transfer of data can be found in the Appendix (see 8.2).

Print Mode: The print mode can be set to either Manual or Auto. At the same time, you must specify through the additional values PF On or PF Off (PF stands for Page Feed), whether the printer should print one page after each print command or only when the page is full (e.g., Auto/PF On).

In the Auto setting, the data are automatically output after every measurement. In the Manual setting, you must press PRINT after a measurement to output the data. In the PF On setting, up to four outputs fi t onto one page. You can print the page at any time in advance with the Page Feed command on your printer.

You can abort parameter input at any time by pressing either ENTER or CANCEL. If you press CANCEL, your entries are discarded and the previous settings are restored. Pressing PRINT will printout all setup parameters for reference purposes. Pressing ENTER gives you the option of saving your new settings in one of the 20 setups.

A warning message will appear if a measurement is shown in the display and you have changed the Sample Volume. You will be reminded that the current measuring result may be lost if you confi rm the message with ENTER. At this point, you can press CANCEL to return to the setup screen. The changes you have made to the measuring parameters will then be reset.



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SAVING SETUP DT-2AB-9999 15.04.2002 10:15

Capillary: 150 μm

Setup No: 00 01 02 03 04 05 06 07 08 09

*

10 11 12 13 14 15 16 17 18 19

USE NEXT TO SELECT SETUP AND PRESS ENTER TO SAVE.USE CANCEL TO WORK WITH NEW PARAMETERS WITHOUT SAVING.

You must save your entries if you want the settings to be available next time you switch on the CASY Counter. To do this, press NEXT to select one of the 20 setups and then press ENTER. The setup currently loaded will be offered as a default setting. You may select both setups that have already been used and tagged with * and also unused setups. If you select a setup tagged with *, its stored settings will be overwritten with your new settings. If you select an unused setup, it will appear tagged with * and will be available for selection next time Select Setup is opened.

If you do not wish to save your settings, press CANCEL. The new settings are used until you select an alternative setup or switch off your CASY. They will then no longer be available. As a setup name for unsaved settings, NOT SAVED appears and the setup number is replaced by two forward slashes //.

If you try to overwrite a logged setup, a warning message will appear. You will be reminded that pressing ENTER will permanently delete this setup’s existing settings. Pressing CANCEL enables you to return to the Saving Setup screen in order to select a free setup for saving.



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MAIN MENU - Select Capillary

MAIN MENU - Select Capillary5.5 The CASY Model DT is designed for counting beads used in combination with 454 Sequencing System. Therefore the system is only equipped with the 150 µm capillary. The Select Capillary option opens the CAP SELECTION screen which displays the 150 µm capillary as the only selectable capillary of the system.

CAP. SELECTION DT-2AB-9999 15.04.2002 10:15

Capillary: 150 μm

*

USE NEXT TO SELECT CAPILLARY AND PRESS ENTER TO LOAD.USE CANCEL TO LEAVE SCREEN.

The system will always use the 150 µm capillary independent from pressing ENTER or CANCEL in the screen above.



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MAIN MENU - Set Date and Time

MAIN MENU - Set Date and Time5.6 Your CASY Counter has an integral Real-Time-Clock. The Set Date and Time function opens the DATE AND TIME screen, in which you can set date and time. You have the option of changing the date display from EU format “DD.MM.YYYY” to US format “MM/DD/YYYY”.

Use the MENU key to move between settings and press NEXT to skip to the individual fi gures used in the date and time.

Use the � and � keys to change the settings. Press ENTER to confi rm your settings or CANCEL to exit the screen without accepting any changes.

DATE AND TIME DT-2AB-9999 15.04.2002 10:15

Date Format: dd.mm.yyyy

Date: 15.04.2002

Time: 10:15:00




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MAIN MENU - System Settings

MAIN MENU - System Settings5.7 System Settings contains settings relevant to the entire system. In the Cursor Arrow Buttons fi eld, you can specify the default settings for manual control of the cursor positions (see 6.9). The Used Setups for Capillary function allows you to delete setups not needed anymore.

The settings for the Step Width and Link Cursors parameters are selected using the MENU key. Use the � and � keys to change the parameters.

SYSTEM SETTINGS DT-2AB-9999 15.04.2002 10:15

Cursor Arrow Buttons

Step Width : 1.0 μm

Link Cursors: Off

Used Setups for Capillary 150 μm

Setup No: 00 01 02 03 04 05 06 07 08 09

* * * *

10 11 12 13 14 15 16 17 18 19

* *


Step Width Indicates the increments in microns by which the cursor should move in the direction of greater or smaller values when the arrow keys on the control panel are pressed (see 6.9). You can set increments of between 0.1 µm and 10 µm.

Link Cursors: This option can be either On or Off. In the On setting, the cursors are moved by pressing the arrow keys on the control panel (see 6.9) together in the required direction. This feature enables you to simply record the size distribution of a sample using the steps specifi ed by Step Width.

To delete redundant setups, use the MENU key to set the input marker in the area called Used Setups for Capillary. The diameter of the active measuring capillary is displayed behind Capillary. Pressing NEXT enables you to shift the input marker between all setups tagged with *. Hold the NEXT key down to move the selection marker backwards. If you wish to delete a setup, press the � key. The setup to be deleted is now tagged with D instead of *. You can cancel your selection at any time by pressing the � key at the relevant position. The D is replaced by *. Follow this procedure to select all the setups you wish to delete.

When you have made all the changes you require, press ENTER to accept the new settings. Pressing CANCEL enables you to exit the screen and return to the old settings.

If you have selected certain setups for deletion, a warning message will appear. You will be reminded that pressing ENTER will permanently delete the setups tagged for deletion. Pressing CANCEL enables you to return to the SYSTEM SETTINGS screen to revise your selection. Deleting the currently selected setup will also delete the current measuring data.



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MAIN MENU - Service Menu

MAIN MENU - Service Menu5.8 The Service Menu contains the Dry Liquid System command you require to shut-down your CASY Model DT properly (see 5.3). It also contains two menu items that enable you to check that the CASY Counter electronics are functioning correctly.

SERVICE MENU DT-2AB-9999 15.04.2002 10:15

* Dry Liquid System

Start Selftest

Generate Testpattern

USE MENU TO SELECT AND PRESS ENTER TO START.USE CANCEL TO LEAVE SCREEN.

Start Selftest performs the self-test known from system start (see 4.3). This serves to monitor the hardware, the pressure system and all memory functions in the CASY Counter (program, calibration, setups). The individual checks are confi rmed by displaying OK. If an error is detected, the corresponding error message will appear (see 8.1).

The Generate Testpattern feature creates a test size distribution and performs a standardized evaluation using specifi ed parameters. You will fi nd a test printout, generated during the fi nal factory check, in your system documentation. This command verifi es that the evaluation electronics function properly and all calculations produced by the evaluation software are correct.

Use the MENU and ENTER keys to select and start the individual commands. Pressing CANCEL enables you to exit the Service Menu.



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Cursor control using the control panel keys

Cursor control using the control panel 5.9 keys

CASY Model DT enables you to change the cursor positions even after a measurement has been performed. This is done using manual cursor control keys on the left-hand side of the control panel. If the three size ranges offered by CASY Model DT are inadequate for your application, you can use this function to get a more detailed analysis of your samples for various size categories. You can also record the entire size distribution of your sample using a predefi ned step width (see 7.5).

Using the L-CURSOR and R-CURSOR keys activate the mode to manually set the cursors. The selected cursor is tagged with *.

SYSTEM SETTINGS DT-2AB-9999 15.04.2002 10:15

Cursor Arrow Buttons

Step Width : : 1.0 μm

Link Cursors: : Off

Actual Cursor Positions

Left Cursor: : * 7.6 μm

Right Cursor: : 12.8 μm

USE MENU AND NEXT TO STEP. USE +/- TO EDIT.USE THE ARROW BUTTONS TO CHANGE CURSOR POSITIONS.USE CANCEL TO LEAVE SCREEN OR ENTER TO CONFIRM CHANGES.

Alternate between L-Cursor and R-Cursor to activate the setting for the left and/or right-hand evaluation cursor. The * is shifted accordingly.

Using the and keys, shift the active cursor in the direction of smaller or greater values. The numeric display of the cursor position is continuously updated. The cursor movement increment is specifi ed by the Step Width parameter. When the screen is opened, this parameter has the default value defi ned in System Settings (see 6.7). The value can however be changed at any time to suit current requirements. Use the MENU key to select the parameter and the NEXT key to select the number to be changed. Change the value using the � and � keys.

You can shift both cursors simultaneously by the Step Width value, if the Link Cursors option is set to On. In this case, both cursors are tagged with *. The default value of this parameter can also be specifi ed in System Settings (see 6.7). It is advisable to shift both cursors simultaneously, e.g., to record a size distribution by a specifi ed increment.

The manual cursor setting mode can be terminated by pressing either ENTER or CANCEL. If you press ENTER, the new cursor positions are accepted as active settings and the calculation of the displayed measured values is updated. If you press CANCEL, the previous cursor position settings are restored. All new changes are rejected.

While you remain in the manual cursor control mode, only the L-Cursor, R-Cursor, , , MENU, NEXT, �, �, CANCEL and ENTER keys are active. You must

terminate manual cursor control before you can use other control panel functions.


35Performing and evaluating measurements

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Preparing samples

Performing and evaluating 6. measurements

Working with your CASY Model DT is very simple. The Counter is ready to start a measurement as soon as it is switched on. However, please observe the following before you start to measure your samples:

� On initial start-up or after moving the Counter, check the points cited in Section 4.1 (Choosing the location) and Section 4.2 (Cabling).

� Follow the steps described in Section 4.4 (Preparing for measurement). Specifi cally:

� Fitting (see 3.2) the required measuring capillary where necessary. � Selecting an appropriate setup (see 6.3) or manually inputting the parameters

(see 6.4). � Background monitoring (see 4.4).

Please note that after power-up, the Counter will automatically load the previously used setup. Further information on how to manually specify parameters or create a new setup can be found in Section 7.5.

Preparing samples6.1 � Fill a CASY cup with 10 ml CASY ton.

� Add an aliquot of the particle suspension to be examined.

� Secure the lid and mix the sample by tilting the CASY cup three times. Carefully avoid the formation of bubbles or foam!

a) Your CASY Counter can measure only the samples you provide. The quality of the measuring result depends on how well you prepare your samples. Standardize the sample preparation procedure as far as possible (used timing of sample preparation, duration and intensity of particle re-suspension, carrying out the required dilution steps etc.).

b) You should always freshly prepare your samples just before the measurement. Depending on the nature of the particles to be analysed, there is no guarantee that they will remain unchanged in CASY ton in terms of count over long periods. If a sample remains in CASY ton for longer than two minutes, you must remix it before the measurement.

c) Always use new CASY cups. This ensures that your measuring results cannot be impaired by high background caused by contaminated measuring cups.



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Measuring samples and displaying the measuring results

Measuring samples and displaying the 6.2 measuring results

� Rotate the recess in the sample platform until it points forwards and remove the CASY

cup from below the measuring capillary.

� Place the sample to be measured below the measuring capillary and ensure that the external platinum electrode is immersed in the sample.

� Rotate the sample platform further to the side.

� Press START to perform a measurement using the current parameters. CASY Model DT automatically works through all the necessary steps. After a short time, the results for your sample appears in the display.

SET : Beads-01│SN : DT-2AB-9999│CAP : 150 μmDATE: 10.11.2009│SVOL: 3 x 400 μl│L.CURSOR: 15.0 μmTIME: 10:15│DIL : 3.333E+03 │R.CURSOR: 25.0 μm

Conc Range: Ok

Counts : 4533

Counts/ml: 3.3 - 15.0 μm: 1.127e+06

Counts/ml: 15.0 - 25.0 μm: 1.140e+07

Counts/ml: 25.0 -100.0 μm: 3.684e+05

This example shows the measuring results for an example of beads with a mean size of 20 µm:

� The size range between 3.3 and 15 µm contains objects too small to be beads mainly debris particles.

� The size range between 15 and 25 µm contains the objects of interest, means the beads with a size of 20µm.

� The size range between 25 and 100 µm contains objects too big to be single beads (partially aggregates).

The example shows the format of the numerical display for the Display Mode = Standard setting. Details on possible settings can be found in Section 6.4. Depending on your settings, some of the parameters not visible in this example may be shown! Depending on your applications, the settings of the cursor positions may differ completely from the ones shown in the example.

The numerical results display is divided into two distinct areas. The header, which is separated by a horizontal line, contains a complete documentation on all measuring parameters used for the measurement. The main section contains the measuring results. This area is divided into two sub-sections. The top section contains the general data for observance of the correct concentration range (see 7.4) and for the total number of recorded count events. Separated by another horizontal line, the bottom section contains the concentrations for the set cursor ranges and any relevant percentages.



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Measuring samples and displaying the measuring results

Measuring parameters in the header of the numerical display

SET: The setup used for the measurement in NAME-## format. If the current setup is not stored, NOT SAVED will appear here.

TIME: The time at which the measurement was performed.

DATE: The date on which the measurement was performed.

SN: The serial number of your CASY Model DT.

SVOL: The sample volume used for the measurement, indicated as cycles × measuring volume.

DIL: The dilution factor used to calculate the sample concentration.

CAP: The diameter of the used measuring capillary.

L.CURSOR: The position of the left cursor used to calculate the measuring result.

R.CURSOR: The position of the right cursor used to calculate the measuring result.

Measuring results

The actual measured sample volume and the entered dilution factor are taken into account in the calculation of all values indicated in ml.

Conc. Range: Indicates whether the measured concentration lies within the permissible range. Possible indications are Ok or Too High. The permissible maximum concentration for the used 150µm capillary is < 20.000 counts/ml (see below).

Counts: Total number of particles counted in the range of the 150 µm capillary.

Counts/ml ###.#-###.# µm:

(1) Concentration of particles in the size range between the lower measuring limit (3,3 µm) and the left cursor.


(2) Concentration of particles in the size range between the left and right cursors. If Display Mode = %Calculation is selected the percentage relative to the Total Counts/ml parameter (Total/ml) is also indicated.


(3) Concentration of particles in the size range between the right cursor and the end of the measuring range of the capillary (100 µm). If Display Mode = %Calculation is selected the percentage relative to the Total Counts/ml parameter (Total/ml) is also indicated.

Total/ml ###.#-###.# µm:

This value is indicated only if the Total Counts/ml is set to On in the current setup. It indicates the concentration of particles in the size range between the left cursor and the end of the measuring range of the capillary (100 µm). It serves as a reference value (100%) for calculating the percentages with Display Mode %Calcualtion.



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Points to be observed when performing measurements

Points to be observed when performing 6.3 measurements

For the measuring operation, use only CASY ton, CASY cups and CASY clean specifi ed for the CASY Counter by Roche Innovatis AG. Using alternative measuring solutions, cups and cleaning agents may lead to incorrect measuring results and cause damage to the CASY Model DT fl uid system. Roche Innovatis AG accepts no liability for damage caused by using alternative consumables.

A smooth measuring operation depends on the Counter undergoing the regular Weekly Cleaning Routine (see 5.1.) using CASY clean (see Section 5.1.)! You must ensure that your CASY Counter is also kept clean between cleaning cycles. Specifi cally that parti-cles never remain in the fl uid system for long periods! The daily background monitoring procedure (see 4.4) and the visual check on the vertical tube will indicate when the Coun-ter is becoming contaminated.

The vertical tube is a calibrated glass pipette through which the CASY Counter, controlled by a light barrier, aspirates the set measuring volume. The vertical tube pipe is visible in the inspection window at the front of the measuring unit.

During the measurement, you can visually track the rising level of the fl uid meniscus. Depending on the level in the vertical tube, one or several light diodes illuminate (Green = start of measurement, Red 1 = 200 µl measuring volume, Red 2 = 400 µl measuring volume). Judge the status of the vertical tube as you would a glass pipette. If you see that the fl uid is no longer running cleanly and drops of fl uid remain suspended on the edge, the calculation of your sample concentration may be adversely affected. In such cases, run a cleaning routine using CASY clean (see 5.1.).

Changing the samples

Provided you measure samples of the same particle type in a similar concentration range, you do not have to fl ush the Counter when the samples are changed. If you expect larger concentration differences or wish to perform measurements with a different particle type, before a new measurement, place a CASY cup containing freshly fi ltered CASY ton below the measuring capillary and fl ush the CASY Counter using the CLEAN cycle.

The Clean cycle is designed to get rid of particles and contaminations in the internal fl uid system or air bubbles in the vertical tube or blocking of the measuring pore. The Clean cycle alone cannot help to get rid of particles and contamination on the outer surface of the capillary and the outer electrode! For that use a CASY

cup with fi ltered CASY ton and fl ush the outer surface of the capillary and the outer electrode.



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Sample concentration

For correct measurement results, the concentration of the diluted particle suspension in the CASY cup should not exceed a certain maximum value. The concentration limit depends on the size of the measuring capillary means for the used 150 µm measuring capillary: < 20.000 counts/ml.

Higher concentrations than specifi ed result in coincidence errors, i.e., an increased probability that two particles will be in the measuring pore at any one time. The CASY Counter will evaluate two particles being in the measuring pore at any one time as if they were one particle with a correspondingly larger volume. The calculated particle count will therefore be too low and the size distribution will shift towards the larger volumes.

The coincidence limits do not depend on the positions of your evaluation cursor settings. They are always determined for the entire physical measuring range of the used measuring capillary (approx. 2% to 80% of the measuring pore diameter). This guarantees that particle count and particle volume are always measured correctly.

You are not required to check yourself whether the concentration of your samples is too high. The CASY Counter displays the following warning message if the coincidence limit values indicated above are exceeded.

ERROR SCREEN DT-2AB-9999 15.04.2002 10:15

Concentration too high !

Please dilute your sample Measured Counts/ml : 34345 Max Counts/ml allowed : 20000

Further information will be Provided by your manual!

USE ENTER TO CONFIRM.PRESS CLEAN TO FLUSH THE LIQUID SYSTEM.

You must confi rm this message by pressing ENTER. The measuring result is displayed, but the ConcRng parameter is shown as Too high. To obtain valid measuring results, you must adequately dilute your sample and repeat the measurement.



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Measuring pore blockages

During the measurement, the CASY Counter exactly monitors the rise times of the fl uid column. This procedure ensures the reliable detection of even fi lm-like deposits (protein, lipids, etc.) in the measuring pore, which shrink rather than actually block the pore, resulting in incorrect volume calculation. If the rise time exceeds a critical limit, the following error message appears:


Measuring Timeout !

Capillary is possibly blocked.

Further information will be provided by your manual.


Most blockages occurring during the measuring operation result from the temporary presence of very large particles in front of the measuring pore, which act like a “valve fl ap”. You can repeat the measurement immediately after confi rming the error message by pressing ENTER. Shaking the CASY cup lightly often helps to remove contamination from the outside of the measuring pore. If the blockage remains, you can usually remove the contamination easily using the CLEAN function.

During the CLEAN cycle, fl uid is fl ushed under pressure from inside the measuring capillary through the measuring pore and into the sample cup. To prevent your sample becoming diluted or contaminated, replace it with a CASY cup containing clean CASY

ton before you run a CLEAN cycle.

If even repeated CLEAN cycles fail to remove a measuring pore blockage, you can unscrew the measuring capillary (see 3.2) and try to fl ush the measuring pore through from the top using a syringe. If necessary, place the measuring capillaries in CASY clean and leave overnight.



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Air bubbles

The CASY Counter precisely monitors the incidence of “critical” air bubbles in the rising fl uid column within the vertical tube. Large air bubbles affect the accuracy of the sample volume measurement and may therefore result in the particle concentration being measured incorrectly. If an air bubble exceeds a critical size during a measurement, the following error message appears:


Large Air Bubble detected !

Regular use of CASYclean. is recommended.

Further information will be

provided by your manual.


If you observe the vertical tube in the inspection window at the front of the measuring stand, you will be able to see the air bubble. If the air bubble rises to the fl uid meniscus and disappears, it will not affect subsequent measurements. You can repeat the measurement immediately after confi rming the error message by pressing ENTER. If, however, the air bubble remains suspended in the vertical tube, you must remove it by replenishing the fl uid system using the CLEAN fl ushing function.

Deposits within the fl uid system sometimes become noticeable through frequent air bubble error messages. If necessary, run a CASY clean cycle (see 5.1) to remedy the problem. En-sure that the fl uid system is rinsed thoroughly with distilled water to remove any remnants of CASY clean!

If, during the measurement, only small air bubbles are evident within the vertical tube, no action is necessary. Small air bubbles – which are higher in number with small measuring capillaries – are caused by the process of electrolysis in the inner electrode. These are quite normal, no error message is triggered and the measurement is not affected.



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Empty Storage container

Should the CASY Counter fail to completely replenish the fl uid system during a CLEAN cycle, the following error message appears:


Error-Code: PR-8

Liquid System could not be fi lled during Clean!

Please check, that there is enough CASYton in the storage container. Further information will be provided by your manual.

PRESS ENTER TO CONFIRM OR PRINT TO GET A HARDCOPYPRESS CLEAN TO FLUSH THE LIQUID SYSTEM.

Ensure that there is suffi cient CASY ton in the storage container (right hand container). Replenish the CASY ton if necessary and run another CLEAN cycle. If the error message re-appears even when the storage container is suffi ciently full, the pressure system may be mal-functioning or the internal tube system is blocked. In this case, contact Roche Innovatis AG.



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Setting the cursor positions

Setting the cursor positions6.4 The system is especially designed to measure beads used for 454 Life Sciences sequencing. The recommended setting for the specifi c beads to be measured is shown below:

SETUP NUMBER: 01 NAME: Beads

Capillary : 150 μmSample Volume : 3 x 400 μl

Dilution : 3.333e+03

Left Cursor : 15.00 μmRight Cursor : 25.00 μm

Display Mode : StandardTotal Counts/ml : Off

Interface : Parallel Print-Mode : Manual/PF On


Setup number, setup name and settings for interface as well as print-mode might be different depending on your requirements, but will not change the measuring results.

In general accurate measuring results rely on the cursor positions being correctly adjusted. It is particularly important for evaluation of the data, that all samples to be compared are measured with identical settings. For new applications it is generally advisable to specify the cursor positions in a setup after the appropriate preliminary tests have been performed. To determine the correct settings, you must record the size distribution of one or several typical samples. The samples must be measured only once. You can then use the manual cursor control keys (see 6.9), in order to scan the recorded data with a predefi ned Step Width (see 6.9). The distance of the cursors must be same as the set Step Width.

Before you can defi ne the cursor positions necessary for your desired application you must record the size distribution of the particles to be analysed. To do this, follow the recommended instructions as described in the next chapter:



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Instructions for recording a size distribution using CASY Model DT:

In most cases, it is not necessary to record the entire size distribution with the smallest possible increments (0.1 µm). To minimise the amount of work, it is advisable to scan the size distribution initially with larger increments (e.g.: 0.5 or 1.0 µm) and then make a fi ne analysis of just the areas of interest (e.g.: increments 0.1 or 0.2 µm). Details on processing the measuring parameters and handling the manual cursor control can be found under 6.4 and 6.9. Proceed as follows:

1 Prepare a sample using a CASY cup fi lled with 10 ml fi ltered CASY ton and an appropriate sample volume of the particles to be analysed to get a recommended concentration between 1.000 and 20.000 particles/ml in the CASY cup. Perform a measurement using at least a measuring volume of 3×400 µl.

2 Following the measurement, press the L-Cursor or R-Cursor key to open the manual cursor control screen (see 6.9.)

3 Set the Step Width to the value you require (e.g., 1.0 µm).

4 Use the and keys to position the two cursors at the start of the area of the size distribution to be scanned. The distance between left and right cursor must be the same as the set Step Width (e.g., 4.0 to 5.0 µm with Step Width 1.0 µm).

5 Set the Link Cursors option to On and press ENTER to confi rm your settings.

6 Record the fi rst result (e.g., Counts/ml for the range 4.0 to 5.0 µm) in the results table (see below).

7 Open the manual cursor control screen again using the L-Cursor or R-Cursor key and press the key once. Both cursors are increased by the value defi ned as the Step Width (e.g., to 5.0 to 6.0 µm).

8 Press ENTER and read off the next result (e.g., Counts/ml for the range 5.0 to 6.0 µm).

9 Repeat steps 7 and 8 until you have reached the end of the size range to be scanned. In case you roughly know the size range of the particles to be analysed, it might be suffi cient to examine the according size range only.

10 Use a spreadsheet software of your choice (e.g., Microsoft Excel) and apply the determined concentration values to a µm scale to obtain a picture of the size distribution (see the example below).

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The ranges of the size distribution important for setting the cursor (minima) can if necessary be examined in more detail following the same procedure, except using a smaller increment. On the basis of the generated size distribution you can defi ne the cursor positions according to your demands.

In the example below the left cursor was placed to 7µm and the right cursor to 14 µm, which means that the size range from 7 to 14 µm is defi ned as the size range containing the objects of interest.



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Size distribution of 10 µm beads recorded in increments of 0,5 µm:

When you have determined the correct settings for your objects to be analysed, you should save them as setup and assign them a typical name. This guarantees that the same objects will always be measured with the same settings, i.e., that the measurements are and remain comparable.



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Outputting the results

Outputting the results6.5 The data can be output via either a parallel or a serial interface. The parallel interface is provided to connect a PCL-capable printer (PCL = Printer Control Language). All Hewlett-Packard printers and compatible products satisfy this requirement (see Appendix 8.3., Technical Data). The data can be transferred to a computer for further editing via the serial interface (see Appendix 8.2, Data output via the serial interface).

If you have set the Print Mode parameter to Auto, the data are automatically output after each measurement via interfaces specifi ed in the current parameters (see 6.4). If Print Mode is set to Manual, you must start to output the measuring results by pressing PRINT.

The numerical output has the following format:

CASY Model DT Serial Number : DT-2AB-9999 Date : 10.11.09

Setup : Beads-01 Time : 10:15

Comment : _________________________________________________________________________________

User/Siganture : _________________________________________________________________________________

Capillary : 150 μm Conc. Range : OK Interface : Parallel

Cursor left : 15.0 μm Counts : 7516 Print Mode : Manual/PF Off

Cursor right : 25.0 μm Counts/ml 3.3- 15.0 μm : 3.347e+06

Sample Volume : 3 x 400 μl Counts/ml 15.0- 25.0 μm : 2.765e+07

Dilution : 3.333e+03 Counts/ml 25.0-100.0 μm : 1.548e+06

Display Mode : Standard

The Comment and User/Signature rows are intended for hand-written comments on the measuring results and a signature where appropriate.

The example shows the format of the output for the Display Mode = Standard setting. Details on possible settings can be found in Section 6.4. Depending on your settings, some of the parameters not visible in this example are maybe printed out! Depending on your applications, the settings of the cursor positions may differ completely from the ones shown in the example.

If you have selected Parallel or P + S as an interface, a printer must be connected to the parallel interface. If you have omitted to switch on your printer or if it is not correctly connected, the following error message appears when you try to output data.


Error-Code: CM-1

Printer does not respond! Be sure that your Printer is switched on and connected.

PRESS ENTER TO CONFIRM OR PRINT TO GET A HARDCOPY

Press ENTER to confi rm the error message, switch the printer on check that it is correctly connected. Having eliminated the cause of the error, you can resume outputting the data by pressing PRINT.


47Appendix

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Error messages

Appendix7.

Error messages7.1 The CASY Counter permanently monitors all its system functions and reports error messages as soon as a fault is detected:


Error-Code: XX-##

Please shut off your CASY For at least 5 seconds. If error persists note the code and contact Schärfe System or your local distributor.

PRESS ENTER TO CONFIRM OR PRINT TO GET A HARDCOPY

Generally, an error code consisting of two letters followed by a hyphen and a one or two fi gure number is output (e.g.: PR-7).

In special cases, a combination of up to 12 hexadecimal characters may be output, which are arranged in 3 blocks separated by colons.

If you receive an errors message, fi rst try to switch off and then restart your CASY Counter. Errors can sometimes be triggered by power supply faults. If no error message appears when you restart the Counter, no further problems should occur. If the same error message appears during the self-test or measuring operation, you should note the error code. You can also output the error message on the connected printer by pressing PRINT.

The following errors may be caused by false handling and can usually be easily cleared.

CM-1: Check that the printer is correctly connected and switched on.

PR-7 Check that the selected capillary setting corresponds to the measuring capillary in use.

PR-8 Check that the storage container is full of CASY ton.

All other error messages that have an error code are caused by faults within the Counter and cannot be cleared. The same applies to the errors listed above, if they cannot be cleared by the actions described. Please contact Service at Roche Innovatis AG or your nearest dealer to clarify further procedure.



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Data output via the serial interface7.2 You can transfer all the data relevant to a measurement via the serial interface to a connected computer. This data includes all the measuring parameters specifi ed in the active settings and all the values calculated from the current cursor settings.

Format of serial data transfer

Row Name of Parameter Format Comments

1 Serial Number @@@@@@@@@@@@

2 Setup Name @@@@@@@@@@

3 Setup Number ## 00 - 19

4 Time ##:## 24 h format

5 Date dd.mm.yyyy oder mm/dd/yyyy depending on the selected format

6 Dilution factor #.###e+##

7 Measuring Cycles ## 1 - 10

8 Sample Volume (µl) ###.## corrected based on volume calibration

9 Display Mode # 0=Standard, 1=%Viability, 2=%Calc.

10 Total Counts/ml Mode # 0=Off, 1=On

11 Capillary Diameter (µm) ###

12 Left Cursor (µm) ###.##

13 Right Cursor (µm) ###.##

14 Counts ########## in total measuring range of capillary

15 Concentration Range # 0=Ok, 1=Too High

16 Counts/ml < CL #.###e+## below the left cursor

17 Counts/ml >CL <CR #.###e+## between left and right cursor

18 Counts/ml >CR #.###e+## above the right cursor

19 Total Counts/ml >CL #.###e+## above the left cursor*

20 %Counts >CL <CR ###.## between left and right cursor*

21 %Counts >CR ###.## above the right cursor*

22 Check Sum XXXXXXXX 4 bytes hexadecimal

Depending on your setup settings, some data tagged with * is output as not calculated.

To enable the CASY Counter to output the measured data via the serial interface, the Interface parameter must be set to Ser or P + S in the current setup. If the Print Mode is set to Auto, the data is automatically exported after every measurement. If Print is set to Manual, the data is output by pressing PRINT on the control panel. The data is exported in text format (ASCII) and the individual values are separated by CR/LF.

To be able to accept the data, your computer requires a free serial interface. The connection is established by a straight serial transfer cable with a minimum of three signal lines (T×D, R×D and SignalGround).

The CASY Counter uses the following Transfer parameters: Baudrate: 19200

Parity: None

Databits: 8

Stopbits: 1

Data output via the serial interface


49Appendix

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Technical data on CASY Model DT7.3

Function: Electronic Counter for determining particle count.

Dimensions: Height: 39 cm

Width: 31 cm

Depth: 39 cm

Weight: 14,5 kg (including control panel)

Ambient conditions: maximum operating temperature:

recommended operating temperature:

10°C to 40°C

15°C to 32°C

Power supply: 230 V~ / 50 Hz

115V~ / 60 Hz

Power consumption: max. 112 VA

Fuses (primary): 230V: 2 × 800 mAT

115V: 2 × 1.6 AT

Fuses (secondary): SI1: T 160mA

SI2: T 1.6A

SI3: T 400mA

SI4: T 2.5A

SI5: T 400mA

Volume measuring section and pressure system:

Calibrated, opto-electronically controlled volume measuring section with no-maintenance, sensor controlled pressure system. Mercury-free.

Measuring principle: Electrical resistance measurement in 1 MHz, low voltage fi eld with pulse area analysis by Schärfe System.

Measuring range: 3.3 µm to 100 µm

Measurement dynamics: Ratio of smallest to largest particles that can be analysed simultaneously. > 1 : 70,000 in terms of volume, > 1 : 40 in terms of diameter

Typical analysis time: 10 sec

Typical sample volume: 2 µl – 100 µl

Measuring volume: 200 µl – 4 ml

Analysis/results: Analysis of the size distribution using two application-specifi c, adjustable cursors:

particle count: absolute, relative -%-,

Interfaces: Serial, RS232 (DB9)

Parallel (DB25)

Only EIC or UL-tested devices can be connected to the RS232 and printer interfaces.

Printer output: PCL (Hewlett- Packard compatible printer)

Printout of: Numerical result

Setups

Error messages

Data export: ASCII

��

Technical data on CASY Model DT



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Technical features: Permanent, highly stable calibration for biological media – certifi ed by Roche Roche Innovatis AG, identical for all CASY systems.

Recording of measuring results, including date, time, setup name, device serial number and all device settings.

System self-test checking all measuring-specifi c parameters before and after the measurement.

Electronic monitoring of the measuring pore.

Automatic fl ushing of the measuring pore.

Automatic detection and display of operating and system errors.

Integrated service functions for fast error diagnosis.

Fanless system suitable for use in clean rooms.

Operation: Menu-driven operation and control of all functions through a movable control panel with membrane keyboard and display.

All application-specifi c device settings can be saved as setup.

20 setups– each assigned a different name.

Output of results to display and printer.

Export function for computers via the serial interface.

Maintenance: If cared for on a regular basis, the CASY Counter is virtually maintenance-free. If a service becomes necessary or requested by the user, contact Roche Innovatis AG. Servicing work may only be carried out by engineers who have been trained and approved by Roche Innovatis AG.

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For life science research only. Not for use in diagnostic procedures.

454, 454 LIFE SCIENCES, 454 SEQUENCING, CASY and INNOVATIS are trademarks of Roche.

Technical data on CASY Model DT


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