LUMAT LB 9507 User manual

LUMAT
LB 9507
Variable Injector
Firmware Version 5.03
Id. No.: 81 957 BA4
Rev. No.: 06 5.03.2001

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LB 9507 Contents
i
Contents
Trademarks iv
1. SYSTEM DESCRIPTION 1-1
1.1 Application 1-1
1.2 Structure and Operation of the Software 1-3
1.3 The Principle of Measurement 1-8
1.4 Sample Tubes 1-9
1.5 Data Management and Software update 1-10
1.6 Simple and Reliable Operation 1-11
1.7 Instrument and software versions 1-11
2. DESIGN OF LUMAT LB 9507 2-1
2.1 Keyboard 2-4
2.2 Display 2-5
2.3 Printer 2-5
2.4 Measurement Unit 2-6
2.5 Reagent Storage Area 2-10
2.6 Variable injectors 2-11
2.7 Reagent Supply 2-13
2.8 Memory Management 2-14
3. GETTING STARTED 3-1
3.1 Instrument Setup 3-1
3.2 Power Supply 3-1
3.3 Checking the Paper Supply 3-1
3.4 Switching on Lumat 3-2
3.5 System Configuration Parameters 3-3
3.5.1 Description of the System Configuration Parameters 3-4
3.5.2 System Configuration Table 3-6
3.6 Instrument Parameters 3-7
3.6.1 Setting/Verifying the Instrument Parameters 3-7
3.6.2 Changing the Instrument Parameters 3-9
3.7 Reagent Supply 3-11
3.8 Safety Precautions for Operation 3-13
4. STRUCTURE AND OPERATION OF SOFTWARE 4-1
4.1 Operation 4-1
4.2 The Six Functions of the Main Menu 4-2
4.2.1 <MEASURE> Menu 4-3
4.2.2 <PROTOCOL> Menu 4-4
4.2.3 <OPERATOR FUNCTIONS> Menu 4-6
4.2.4 <SYSTEM CONFIGURATION> Menu 4-7
4.2.5 <INSTRUMENT PARAMETER> Menu 4-8
4.2.6 <TURN ONCE> function 4-8
4.2.7 Service functions 4-8
4.3 General Procedure 4-9
5. RAW DATA MEASUREMENTS 5-1

Contents LB 9507
ii
5.1 Introduction 5-1
5.2 Creating Raw Data Protocols 5-3
5.3 Editing/Deleting Protocols 5-9
5.4 Performing Raw Data Measurements 5-11
6. CUT-OFF MEASUREMENTS 6-1
6.1 Introduction 6-1
6.2 Creating CUT-OFF Protocols 6-4
6.3 Editing/Deleting Protocols 6-12
6.4 Running Cut-off Measurements 6-14
7. QUANTITATIVE MEASUREMENTS (LIA...) 7-1
7.1 Introduction 7-1
7.2 Setting Up Quantitative Protocols 7-3
7.3 Editing/Deleting Protocols 7-12
7.4 Entering a Master Curve 7-14
7.5 Performing Quantitative Measurements 7-17
7.5.1 Measurement with a Complete Standard Curve 7-17
7.5.2 Measurements with Master Curve 7-25
8. T-UPTAKE/FTI-MEASUREMENTS 8-1
8.1 Introduction 8-1
8.2 Creating T-uptake/FTI protocols 8-3
8.3 Editing/Deleting protocols 8-12
8.4 Performing T-uptake/FTI Measurements 8-14
9. DUAL-LUCIFERASETM REPORTER GENE ASSAY 9-1
9.1 Creating Protocols for Dual-LuciferaseTM Assay 9-3
9.1.1 With 2 injectors installed 9-3
9.1.2 With 1 injector installed 9-8
9.1.3 Without injectors 9-11
9.2 Editing/Deleting Protocols 9-14
9.3 Performing Dual-LuciferaseTM Reporter Gene Assay
Measurements 9-16
10. KINETIC AND REPEATED MEASUREMENTS 10-1
10.1 Kinetic Option in Raw Data Protocol 10-1
10.2 Repeated Measurements 10-2
10.2.1 Creating Repeated Protocols 10-4
10.2.2 Editing/Deleting Protocols 10-9
10.2.3 Performing Repeated Measurements 10-11
10.3 Ratemeter 10-14
11. OPERATOR FUNCTIONS 11-1
11.1 <REAGENT> Function 11-2
11.1.1 Loading reagents 11-3
11.1.2 Refreshing reagents 11-4
11.1.3 Washing of the injector system 11-5
11.1.4 Removing reagents 11-7
11.2 Performance test 11-8
11.3 Manual Data Entry 11-9

LB 9507 Contents
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11.4 Retrieval of Stored Data 11-10
11.4.1 Rerun of a Full Standard Protocol 11-11
11.4.2 Rerun of a 2-Point Calibrator Protocol 11-14
12. CONNECTION TO EXTERNAL COMPUTERS 12-1
12.1 General 12-1
12.2 RS 232 Data Output LB 9507 in Protocol-Specific
Measuring Mode 12-2
12.3 Data Output in Raw Data Measurements 12-4
12.4 Data Output after Manual Input of RLU Values 12-4
12.5 Data Output of Stored Assay Data 12-4
12.6 Data Output of RLU/s in the Ratemeter Mode 12-4
13. MAINTENANCE 13-1
13.1 Supplying Paper 13-1
13.2 Replacing the Mains Fuse 13-2
13.3 Changing the Operating Voltage 13-3
13.4 Cleaning the Injector System 13-3
13.5 Cleaning Lumat 13-3
13.6 Changing the Air Filter 13-3
13.7 Checking the Hose Connections 13-3
14. TROUBLESHOOTING 14-1
14.1 Error Messages during Operation 14-1
14.2 Injector System Malfunction 14-1
14.3 Other Errors 14-2
14.4 Default Values 14-2
15. TECHNICAL SPECIFICATIONS OF LB 9507 15-1
16. FLOW DIAGRAM OF THE MEASUREMENT
PROCEDURE 16-1
APPENDIX 1
A.1 Mathematical Basis 2
A.1.1 Overview of the Mathematical Data Reduction 2
A.1.2 Variable Zero Concentration Factor 4
A.1.3 Spline Function 5
A.1.4 Calculation of a Variable Reference Value 6
for Standardization of the Logit Transformation 6
A.1.5 Extrapolation of the Concentration 7
A.2 Data Acquisition of the Lumat LB 9507 8
via the Serial Interface (I/O-Port) and 8
Saving this Data in a MS-DOS ASCII-File 8
A.3 Connection Cable Lumat/Apple Macintosh Computer 9
A.4 Background measurement 10
A.5 Overload Detection 12
A.6 Glossary 13
A.7 Index I

Contents LB 9507
iv
Trademarks
Dual-Luciferase and Stop and Glow are trademarks of Promega Corp.
Lumat is a trademark of Berthold Technologies.
Macintosh is a registered trademark of Apple Computer Inc.
Excel is a registered trademark of Microsoft Corp.

LB 9507 Preface
I
Preface
Use and Function The Lumat LB 9507 is a semi-automatic luminometer for universal
use in bio- and chemiluminescence, and is very easy to operate. It
can work with up to 2 reagent injectors, and its rotating sampling
method enables samples to be measured and loaded at the same
time.
Special Instructions
and Precautions The manufacturer has undertaken everything to ensure the safe
operation of the instrument (regarding electricity, electronics and
mechanics). The user has to make sure that the equipment is
set up and installed properly to guarantee safe operation:
•Use grounded mains.
•Do not open the instrument.
•Disconnect Lumat from the mains before cleaning.
•If the operational safety of the instrument is no longer ensured,
turn it off and disconnect it from the mains.
•If liquid gets into the instrument, disconnect it from the mains.
•Protect yourself from electrostatic charging (e.g. carpets),
since discharging may damage sensitive electronic
components
•Set up the instrument in a dry room only.
•Be careful when handling strong alkaline or acidic reagents.
Make sure you follow the reagent manufacturer’s instructions.
•If there is a leak in the reagent tubing system you must abort
the measurement series and fix the leak. If necessary, call the
manufacturer’s Technical Service. If a leak occurs there is a
risk of reagent entering the inside of the instrument.
•Only technicians authorized by Berthold should carry out
repairs or modifications on the instrument.
The Lumat LB 9507 should only be used to perform the
functions described in this manual.
The proper use and handling of the instrument requires that the
operating personnel be familiar with this manual. Please read it
carefully before taking the instrument into use - even though the
operation of Lumat may appear simple.

Preface LB 9507
II
The tests and maintenance routines recommended by the
manufacturer should be carried out to ensure the operator’s safety
as well as the correct performance of the instrument.
All maintenance and service routines above and beyond those
mentioned in this Operator’s Manual must only be performed by
service technicians authorized by BERTHOLD TECHNOLOGIES
or by the company itself.
The Structure of
the operating manual Because the Lumat LB 9507 is an universal luminometer, it is
capable of a wide range of applications and different
measurement modes (raw data, cut-off, kinetics, repeated, T-
Uptake/FTI, Dual-LuciferaseTM reporter gene assay and
quantitative measurements). To make it easy for the user who is
working with only one measurement mode to find the required
information quickly, this operating manual includes all the relevant
information on each measurement mode in separate chapters.
This means you will not need to search through the manual to find
a different measurement mode.
The description of each measurement mode follows the same
pattern: first a brief introduction, then a flow chart providing a
summary of the procedure, and finally a detailed explanation of
how to create a protocol and run a measurement, using all the
messages shown on the instrument’s display.
Chapter 1 describes the system construction, the ways it can be used, and
the operating procedure
Chapter 2 provides detailed information on the individual system
components
Chapter 3 describes how to commission Lumat, from assembly to
configuration
Chapter 4 explains the structure and handling of the software and its six
menus
Chapter 5 describes the Raw data measurement mode
Chapter 6 describes the Cut-off measurement mode
Chapter 7 describes the Quantitative measurement mode (LIA..)
Chapter 8 describes the T-Uptake/FTI measurement mode
Chapter 9 describes the Dual-LuciferaseTM Reporter Gene Assay mode
Chapter 10 describes the Kinetic and Repeated measurement mode

LB 9507 Preface
III
Chapter 11 describes the special instrument functions such as washing,
pumping back reagents, performance testing, etc.
Chapter 12 explains how to connect an external PC and how to transfer data
Chapter 13 summarizes the maintenance and service routines that can and
should be carried out by the user
Chapter 14 lists all possible error messages and tells you how to eliminate
problems
Chapter 15 lists technical specifications of Lumat
Chapter 16 contains a diagram and a flow chart to help you find your way
around the menu structure in the Lumat software. The diagram
shows the assignment of the functions to the menus, and the flow
chart shows the procedure when creating measurement protocols
Appendix includes a description of the mathematical basis for calculations, a
glossary and an index to help you find your way around, and finally
the data sheets.
Writing conventions The description of the software and dialogs are based on a writing
convention, which uses repeated symbols and makes reading
easier.
The software dialog, i.e. the input sequences performed by the
user via keyboard and display, are described
a) by illustrating the displays one after another
b) by explanatory texts.
How displays are shown In the displays shown, the functions in bold face are those that
can be selected using softkeys to move to the next display. The
text and numerical entries are only examples, and are also shown
in bold face on the displays illustrated.
This allows you to see the difference immediately between things
defined by the program and individual entries.
Explanatory text In the explanatory text, the menus and functions of the program
and the function keys are shown in pointed brackets <> and bold
face. Text and numerical inputs are placed in inverted commas
and printed in bold face. Actions that the user needs to perform
are immediately recognizable, e.g. enter ”15” and <enter>.

Preface LB 9507
IV
Display numbering For reasons of clarity, the displays within one measurement type
are numbered. In addition, every measurement type and special
function is given an identifying letter. This allows every display to
be easily identified and immediately assigned to a certain
procedure. The following identifying letters are used:
R = Raw data measurements (Chapter 5)
C = Cut-off measurements (Chapter 6)
L = Quantitative (LIA...) measurements (Chapter 7)
T = T-uptake/FTI measurements (Chapter 8)
D = Dual-LuciferaseTM Reporter Gene Assay (Chapter 9)
K = Kinetic measurements (Chapter 10)
W = Washing function (Chapter 11.1)
S = Call up stored data (Chapter 11.4)

LB 9507 Chapter 1: System Description
1-1
1. System Description
1.1 Application The Lumat LB 9507 is a semi-automatic, user-friendly
luminometer for universal use in bio- and chemiluminescence.
Equipped with up to 2 reagent injectors, a highly sensitive
photomultiplier, flexible software and a highly advanced, easy-to-
handle mechanical system, Lumat is excellently suited to
applications such as ATP measurement, immunoassays, DNA
probe assays, calcium measurements and reporter gene assays.
Qualitative classification of unknown samples using the ”cut-off
method” can also be performed.
A specially developed rotating sample holder and transport
system for 2 test tubes allows samples to be measured and
loaded in parallel. This almost doubles the sample throughput
during the normal measurement duration of 5 to 10 seconds, thus
equalling the speeds reached by automatic systems.
The Lumat software has been tailored to the user’s specific needs
and the processes of luminescence measurement. Essentially, it
is divided into 6 basic parts:
1. Raw data measurement
In this measurement mode, the quantity of light measured in RLU
units over a user-defined measuring time is produced for each
sample. The mean value and the coefficient of variation are
generated for replicate groups. You have the option of printing out
a high resolution (up to 2000 data points) curve and 20 data
points with time scale for each sample, which enables you to
analyze the kinetic behaviour of the light emission of a sample.
This measurement mode, which allows up to 2 injections per
sample, is mainly used for ATP measurements or for measuring
luminescence reporter gene assays.
2. Cut-off measurements
Using negative and positive standards, unknown samples are
classified quantitatively by interpreting the results of the
measurements on the basis of predefined RLU limits.
3. Quantitative measurements (LIA, ILMA...)
Known standard concentrations are measured before the
unknown samples. After transformation of the calibration curve
(either log-log or logit-log), the program interpolates standards
and unknown samples. Instead of using the standard
concentrations, you can also enter the values from a master curve
which is defined by the reagent manufacturer.

Chapterl 1: System Description LB 9507
1-2
4. Uptake/FTI measurements
In this measurement mode, a standard is used to convert patient
samples into a %-uptake and then into an FTI-value (Free Thyroid
Index).
5. Dual-LuciferaseTM Reporter Gene Assay
This measurement is used for automatic measurement of
Promega's Dual-LuciferaseTM Reporter Gene Assay (injection-
measurement-injection-measurement). In this measurement
mode, the quantity of light measured in RLU units over a user-
defined measuring time is produced for both measurements of
each sample. The mean value and the coefficient of variation are
generated for replicate groups. The measured data of both
measurements will be evaluated to get the normalized response.
6. Kinetic and Repeated Measurements
These measurement modes are used to follow light emission from
the sample in time. A kinetic protocol can be used to take up to
2000 data points from one measurement. During a repeated
measurement the sample will be measured several times. Each
measurement results in one measurement point.

LB 9507 Chapter 1: System Description
1-3
1.2 Structure and Operation of the Software
The Lumat software is designed for the applications and
processes found in luminescence measurement. In addition to the
five measurement modes, we also distinguish between routine
and single measurements. Kinetic measurements can also be
performed with Lumat.
Routine measurements
Measurement protocols Measurement protocols defining the parameters for measurement
and evaluation are generally set up once for routine
measurements. To run a measurement, one simply uses an
existing protocol and starts the measurement.
Lumat can store up to 40 different measurement protocols. When
creating a protocol, select one of the measurement types
described above and the entry sequence for this measurement
type will appear on the instrument display. Each entry is
automatically printed out on the integrated thermal printer, so that
each protocol is documented.
Once you have set up a measurement protocol with the
parameters you need, you can use it as often as you choose for
later measurements.
Measurements Measurements based on these stored protocols can be performed
by simply selecting one of them and starting the measurement. In
accordance with the instructions defined in the measurement
protocol, instructions for the operations that are expected of the
user and the sequence of tubes being measured are displayed on
the LCD.
Printout At the start of the measurement, all essential parameters are
printed out, followed by the results of the measurement obtained
by the calculation methods defined by the selected measurement
protocol.
Single measurements
You may often want to try out new parameters instead of the
stored protocols. If this is the case, the Raw data measurement
type allows you to enter measurement parameters immediately
before starting a measurement, and then start the measurement.
The most recently used parameter set will remain stored between
individual measurements, allowing for easy correction.

Chapterl 1: System Description LB 9507
1-4
Kinetic measurements
There are three ways to perform kinetic measurements:
1. Using a “RAW DATA" measurement protocol or a "Direct
Entry” measurement which is not stored in the instrument
memory. This kind of measurement provides always a fixed
measurement time. The results can be printed out, or
received on the RS 232 port of a computer. The Berthold
"WinTerm" program is suitable to receive and transfere the
results into Microsoft Excel.
2. Using a “REPEATED” measurement protocol to set up the
injector and timing intervals in combination with a stored
protocol. The results can be printed out, or received on the
RS 232 port of a computer. The Berthold "WinTerm" program
is suitable to receive and transfer the results into Microsoft
Excel.
3. Using continuous measurement in “RATEMETER” mode.In
this mode results are continuously produced until the
measurement is stopped by the user. Results can be printed
out, or received on the RS 232 port of a computer.The
Berthold "WinTerm" program is suitable to receive the results
and transfer them into Microsoft Excel.
The measurement procedure
The measurement procedure is defined by the user in
measurement protocols. The integrated software controls the
measurement and evaluation steps, and displays messages and
instructions for the user. Lumat is a semi-automatic system that
requires only a few simple actions by the operator; this is
supported by an ergonomically sensible arrangement of controls
on the front of the instrument.
To perform a measurement you must observe a simple procedure
which is controlled by the software. If you do not follow this
procedure, error messages will be produced accordingly.
The sample chamber and the sample holder form a single unit.
The sample holder contains 2 holes, each for one sample tube,
and sits on the round measurement chamber as a rotating block.
It is turned in 180° steps by the integrated software, moving one
hole in front of the photomultiplier (measurement position) and the
other into the loading position (see Fig. 1-1).

LB 9507 Chapter 1: System Description
1-5
Fig. 1-1: Measurement unit
In the Lumat’s standby mode, the cap should be closed to
protect the measurement chamber against dust. You can leave it
open during measurement so that tubes can be swapped without
interruption; the measurement chamber into which the tubes are
inserted is sealed against the entry of light anyway. The cap only
needs to be closed if you are measuring samples whose
fluorescent light fades before measurement.
At the start of a measurement, load the 1st tube into the
accessible hole (loading position) as instructed on the display, and
press the <start> button to the right of the measurement
chamber. When the system detects that a tube is loaded, the
measurement chamber rotor will turn through 180° and the tube
will end up in front of the photomultiplier. The measurement
procedure defined in the measurement protocol begins
automatically in this position. The 2nd hole is now in the loading
position on the side facing the user. Load another tube during the
measurement, and press the <start> button once again. The rotor
will not turn until the sample in front of the photomultiplier has
been fully measured. The measured sample tube is transported
back to the loading position and can be replaced while the 2nd
tube is being measured, and so on.
tube
transport
rotor
Loading position
Mirrored measurement
chamber
Spring pin for lifting
the tube in loading
position
Injector head
Injector tips
Measurement
position
Meas. chamber opening
for photomultiplier
Measurement chamber cap Injector lines to the pumps

Chapterl 1: System Description LB 9507
1-6
The program runs through the individual steps for each sample
in accordance with the settings in the measurement protocol:
background measurement, injections, measurement.
Upon completion of the background measurement, the pumps are
activated one after the other. If required, pump # 1 injects a
variable amount of the first reagent into the sample tube. After a
defined delay, pump # 2 also injects a variable amount (which
may differ from the 1st injection) of the second reagent into the
sample tube. Of course, measurements without or with only one
injection are also possible.
The injection of the 2nd reagent typically initiates the chemical
reaction resulting in the emission of light photons. The number of
photons that are emitted during this reaction is indirectly related to
the analyte concentration in the sample.
The photons hitting the cathode of the photomultiplier are
transformed into electrons and amplified as single pulses. A
threshold discriminator suppresses the noise signals of the
photomultiplier. The computer counts the pulses and supplies the
integral result of the light signal (photon counting method).
Every measurement step is automatically documented on the
thermal printer together with the measurement results obtained.

LB 9507 Chapter 1: System Description
1-7
Chart of measurement procedure
Display User action Instrument action
1 SAMPLE 1 TUBE 1
INSERT TUBE... Load the 1st tube
2 SAMPLE 1 TUBE 1
START Press <start> button Rotor turns the 1st tube
in measurement position
3 MEASURING... Measuring tube 1
4 MEASUREMENT (RLU) 133 Display results
5 SAMPLE 2 TUBE 2
INSERT TUBE... Load the 2nd tube
6 SAMPLE 2 TUBE 2
START Press <start> button Rotor turns the 2nd tube
in measurement position
7 MEASURING... Measuring tube 2
8 MEASUREMENT (RLU) 137
REMOVE TUBE Remove the 1st tube Displays results
9 SAMPLE 3 TUBE 3
INSERT TUBE... Load the 3rd tube
10 SAMPLE 3 TUBE 3
START Press <start> button Rotor turns the 3rd tube
in measurement position
Pressing the <exit> button finishes measurement.

Chapterl 1: System Description LB 9507
1-8
1.3 The Principle of Measurement
The emitted light is measured by a selected high-sensitivity, low-
noise photomultiplier. Its spectral sensitivity covers a range
between 390 and 620 nm. All well-established applications in bio-
and chemiluminescence emit in this wavelength range.
The photomultiplier operates as an ultra-fast photon counter. The
photoelectrons released from the photocathode by the light
quanta are multiplied via a dynode chain and trigger a fast pulse
with a rise time of a few nanoseconds at the anode. These pulses
are then amplified by a very fast amplifier. A threshold
discriminator suppresses the low-energy pulses caused by the
noise of the photomultiplier. The single pulses are counted
digitally, their total number being directly proportional to the
emitted quantity of light.
However, the ”relative light units” (= RLU) are used as unit of
measurement for the raw data, and not the number of pulses. The
number of RLUs is found by dividing the directly counted pulses
by ten; moreover, the raw data are multiplied by the RLU-factor
(see Instrument Parameters) which allows compensation of the
inevitable individual fluctuations of the cathode sensitivity of
various photomultipliers.
The kinetics of many chemiluminescence reactions is so fast that
typical counting times are in the range of 1-5 s per measurement.
Thus, sample throughputs of 600 samples per hour are possible.
Bioluminescent samples usually require longer measurement
times.
The individual measurement time can be found in method
publications or in your kit description.

LB 9507 Chapter 1: System Description
1-9
1.4 Sample Tubes The Lumat LB 9507 uses sample tubes with the nominal
dimensions 12 ∅x 75 mm length with round bottoms. However,
due to the special requirements of luminescence measurements
not every type of sample tube can be used.
The permissible diameter variation is limited to a range between
11.5 and 12.1 mm; the length of the tubes may vary from 75 to 76
mm.
Basically, all materials with good transparency, such as
polystyrene, glass and polyethylene may be used. However, some
tube types show a higher and, moreover, fluctuating background
caused by electrostatic charging and phosphorescence.
To prevent electrostatic charging we recommend using sample
tubes with antistatic additives. These can be purchased from
Berthold Technologies.
Phosphorescence can be avoided by not exposing the tubes to
short-wave light prior to measurement. In general, there is less
interference if the tubes are moistened prior to the measurement,
for example during the preceding sample preparation. Never
change the tube type within an assay!

Chapterl 1: System Description LB 9507
1-10
1.5 Data Management and Software update
The Lumat LB 9507 Software is based on two parts:
❑The kernel provides all the basic or elementary functions to
control all the electronic functions, also the download
manager and serial number.
❑The User interface which controls the user functions and all
individual parameters eg. individual measurement times and
measurement methods, reagent timing, data management
for results and set up of evaluation procedures.
This arrangement allows you to load updates and upgrades with
the minimum of expense.
If a power failure occurs, the measured data from the last 30
measurement runs remain stored in a battery-backed memory for
at least 3 months.
The measurement protocols are stored in the instrument’s current
and battery independent internal memory, and can only be lost if
the CPU develops a fault or during a program is update.
A RS232 interface allows the kernel or operating software to be
loaded from a PC to Lumat.
The Update software will be provided only from Berthold
Technologies directly or from by Berthold Technologies authorised
service organisations.