LUMAT LB 9507 Instruction

LUMAT

LB 9507

Variable Injector

Firmware Version 5.03

Id. No.: 81 957 BA4
Rev. No.: 06 5.03.2001

ii

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-LUCIFERASE

TM

REPORTER GENE ASSAY 9-1

9.1 Creating Protocols for Dual-Luciferase

TM

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-Luciferase

TM

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

iii

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 Lum at 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 measur ed and loaded at the sam e
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 t o make sure that the eq uipment 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 ensur ed,

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 necess ary, 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 r equires that the
operating personnel be familiar with this manual. Please read it
carefully before taking the instrum ent 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 mus t 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, T­Uptake/FTI, Dual-Luciferase

TM

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 quick ly, this operating m anual inc ludes all the relevant
information on each measurement mode in separate chapters.
This means you will not need to search through the m anual to f ind
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-Luciferase

TM

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 sof tware and dialogs are bas ed 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 exam ples, and ar e als o 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 explanator y 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 comm as
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 measurem ent 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-Luciferase

TM

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-to­handle mechanical system, Lumat is excellently suited to
applications such as ATP measurement, immunoassays, DNA
probe assays, calcium meas urements 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 lumines cence measurement. Essentially, it
is divided into 6 basic parts:

1. Raw data measurement

In this measurement m ode, 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 f or 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 c ur ve
which is defined by the reagent manufacturer.

Chapterl 1: System Description LB 9507

1-2

4. Uptake/FTI measurements

In this measurem ent mode, a standard is used to convert patient
samples into a %-uptake and then into an F T I- value (F ree Thyroid
Index).

5. Dual-Luciferase

TM

Reporter Gene Assay

This measurement is used for automatic measurement of
Promega's Dual-Luciferase

TM

Reporter Gene Assay (injection­measurement-injection-measurement). In this measurement
mode, the quantity of light measured in RLU units over a user­defined measuring time is produced for both measurements of
each sample. The m ean 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 tak e up to
2000 data points from one measurement. During a repeated
measurement the s ample 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 lum inesc enc e 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 def ining the param eters for m easur em ent

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 diff erent meas urem ent 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 m easurem ent. In
accordance with the instructions defined in the measurement
protocol, instructions for the oper ations that are expected of the
user and the sequence of tubes being m easured are displayed on
the LCD.

Printout At the start of the measurement, all essential parameters are

printed out, followed by the results of the measurem ent 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 measuremen t
type allows you to enter measurement parameters immediately
before starting a measur ement, and then start the measurement.
The most recently used param eter set will rem ain 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 k ind 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 pr otocol 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. T he 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 "WinT erm" 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 s teps, and displays messages and
instructions for the user. Lum at 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 obser ve a s imple 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 cham ber 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 measurem ent chamber against dust. You can leave it
open during measurement s o that tubes can be swapped without
interruption; the measurement c hamber 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 cham ber 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 pres s the <start> button onc e 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 sam ple
in accordance with the settings in the measurement protocol:

background measurement, injections, measurement.

Upon completion of the bac kground meas urement, the pum ps ar e
activated one after the other. If required, pump # 1 injects a
variable amount of the first r eagent 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 cours e, 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 num ber 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 c ounts 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 m easured by a selected high-sensitivity, low­noise photomultiplier. Its spectral sensitivity covers a range
between 390 and 620 nm. All well-established applications in bio­and chemiluminescence emit in this wavelength range.

The photomultiplier operates as an ultr a-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. T hese 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 f or the r aw data, and not the num ber of puls es. The
number of RLUs is f ound by dividing the directly counted pulses
by ten; moreover, the raw data are m ultiplied 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 chemilum inescence reactions is so fast that
typical counting times are in the range of 1-5 s per m easurem ent.
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 luminesc ence measurements
not every type of sample tube can be used.

The permissible diameter variation is lim ited 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 back ground
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 m oistened prior to the m easurement,
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 pr ovides 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 us er functions and all

individual parameters eg. individual measurem ent 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 stor ed in a battery-backed mem ory for
at least 3 months.

The measurem ent protoc ols are s tored in the instr um ent’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 s oftware 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.

LB 9507 Chapter 1: System Description

1-11

1.6 Simple and Reliable Operation

A characteristic feature of the LB 9507 is its user-friendliness.
Possible alternatives in a specific situation ar e displayed in plain
language - either German, English or F rench - as chosen by the
user. You just have to press the ”softkey” below the respective
option to execute this function.

Numerous self-diagnos tic and service functions prevent incorrect
measurements. For example:

➤ Background measurement prior to the injection to detect

unacceptable phosphorescence.

➤ Sample tube detection to rule out injections into the empty

measurement chamber.

➤ Automatic wash cycle to fill the injector system with fresh

reagents if the luminometer has not been used for a long
period of time.

➤ Automatic printout of all entries and results.

➤ Measurement and printout of the kinetics, so you can

determine the best counting time. The k inetics monitor is also
of importance in designing new assays.

1.7 Instrument and software versions

Order number

Lumat LB 9507-0
without injectors

90V – 240V 81957-50

Lumat LB 9507-1
one variable volume injector

90V – 240V 81957-51

Lumat LB 9507-2
two variable volume injectors

90V – 240V 81957-52

Lumat LB 9507-LIA

90V – 240V 81957-53

Please notice that not all the instrument vers ions comprise exactly
the same software environment described in this manual. The
standard version of Lumat LB 9507 does not include the LIA
software module.

LB 9507 Chapter 2: Design of the Lumat

2-1

2. Design of Lumat LB 9507

This chapter describes the various components of Lumat
LB 9507.

Front view

1 Keyboard
2 Display
3 Thermal printer
4 Paper advance button
5 Reagent supply bottle (optional)
6 Injector line (optional)
7 Injector pumps (optional)
8 Sample holder cap (keeps out dust)
9 Measurement chamber with sample holder
10 <start> button

23 45678

1910

Fig. 2-1: Lumat LB 9507 (front view)

Chapter 2: Design of the Lumat LB 9507

2-2

Inside view

Main switch; fuse holder Contrast for printer Host I /O HV; amplifier Air filter Injector pumps
Combi element and display RS 232 and analyzer

Power supply CPU Auxiliary board Stepper m ot or Tube detector Measurement chamber

for turning the

tube transport rotor

Fig. 2-2: Inside view of Lumat

Do not open the instrument!

The Lumat LB 9507 must only be opened by service
technicians authorised by Berthold Technologies!

Injector lines

Photomultiplier

Injector head

LB 9507 Chapter 2: Design of the Lumat

2-3

Rear side The rear panel of Lumat:

1 Mains connection (115 or 230 V)
2 Fuse with selector for voltage, display for selecting voltage
3 Power switch
4 Computer port (I/O port)
5 Printer contrast adjust
6 Display contrast adjust

Air filter Power switch Fuse

0

Host I/O port Display Mains plug

RS 232 Print er

Fig. 2-3: Rear view of Lumat

Chapter 2: Design of the Lumat LB 9507

2-4

2.1 Keyboard

The Lumat functions ar e directed through keyboard entries. The
keyboard consists of twenty-six letter keys, twelve numerical keys,
three softkeys, and five additional function keys (<enter>,
<delete>, <last>, <exit> and <space>). Softkeys are keys with
changing functions defined by software and shown on the display.
The functions are selected using the corresponding buttons
beneath the display.

The keyboard design utilizes pushbuttons which are all sealed to
protect them from liquid spillage (Fig. 2- 4). Audio feedback of key
action is provided by a short beep for correct entries and a longer
beep for incorrect entries.

1 Alphanumeric keyboard
2<space> key
3 Function keys <exit>, <delete>, <last> and <enter>
4 Softkeys
5 Display
6 Softkey functions: left, center, right

6

exit delete last enter

1 2 3 4 5 6 7 8 9 0 .

Q W E R T Y U I O P

A S D F G H J K L -

space Z X C V B N M /

21

Fig. 2-4: Keyboard

5

4

3

LB 9507 Chapter 2: Design of the Lumat

2-5

2.2 Display

The display located above the keyboard is a two-line LCD display,
each line containing 40 characters. T he LED background lighting
switches on when Lumat is powered up.

Functions:

➤ Entries request and display.
➤ T he second line displays the functions of the sof tkeys, which

are activated using the buttons located directly below the
display field

➤ Results display
➤ Instrument status display
➤ User guidance during protocol entry and measurement

2.3 Printer

The thermal printer located on the top left corner of the ins tru ment
has the following functions:

➤➤➤➤ to print the protocols entered
➤➤➤➤ to print the parameters of an individual protocol
➤➤➤➤ to print measurement results and cut-off classes
➤➤➤➤ to plot kinetics curves

As an alternative to the thermal printer you can also connect a PC
to the RS 232 Interface to receive the measured data. A suitable
software is "WinTerm", available from Berthold Technologies.

Chapter 2: Design of the Lumat LB 9507

2-6

2.4 Measurement Unit

The measurem ent unit is located to the right of the keyboard (see
Fig. 2-1, [9]). The sample c hamber is protected agains t dust by a
semi-circular cap. The measurement chamber is located in the
instrument itself and c an only be accessed by removing the cover
of the instrument (see Fig. 2-2).

Structure and function The following diagrams (F igs. 2- 5 to 2- 8) ex plain the s truc tur e and

function of the measurement unit.

The measurement unit comprises the following components:

●

●●

● a black, light-proof measurement chamber casing

●

●●

● the tube transport rotor inside this casing, incorporating 2 holes

for holding sample tubes and a mirrored measurement
chamber

●

●●

● a tube detector

●

●●

● a stepping motor for rotating the rotor

●

●●

● an injector head in the measurement unit lid

●

●●

● an opening for the photomultiplier at the rear.

Injector lines to the pumps

Measurement chamber cap

Fig. 2-5: The structure of the measurement unit (schematic illustration)

Loading position

Mirrored measurement

chamber

Spring pin for lifting
the tube in loading
position

Injector head

Injector tips

Measurement
position

Meas. chamber opening
for

p

hotomultiplier

tube

transport

rotor

LB 9507 Chapter 2: Design of the Lumat

2-7

Tube transport rotor The tube transport rotor is m ade of a special conductive plastic

which prevents electrostatic charges from building up.

The rotor contains two holes positioned opposite each other, and
each can hold a sample tube, m eaning that one hole is always in

loading position (at the front, facing the user) and one in
measurement position. Pressing a button (<START> when

measuring, or the softkey <TURN ONCE>) causes the rotor to
turn through 180°, thus switching the positions of the tubes. The
tube in the loading position is lifted by a spring-pin so that it is
easier to take out (see Figs. 2-5 and 2-6) . This rotation principle

allows samples to be loaded and measured at the same time.

Sealing lips (spring-loaded)

Fig. 2-6: Tube transport rotor

To distinguish between the two measurement chambers, one
of them is marked with a red spot. This measurement
chamber is called the "red" chamber, the oth er is called the
"black" chamber.

The top of the tube transport rotor includes two spring-loaded
sealing lips which shield the measurement chamber from light.

Tube in
measurement
position

Tube in
loading position
(pushed up)

Measurement chamber:
the rear surface
is mirrored to
increase the
light again

Spring pin:
the tube sits on
the spring pin; in the
loading position it is ­ pushed up by a
sloping base

Chapter 2: Design of the Lumat LB 9507

2-8

In case of need the rotor can also be turned manually (see below),
so that it can be placed in positions other than the exact
measurement positions.

The software-controlled rotation movement positions the rotor
exactly; this is vital for measur ement as all of the sam ple m ust be
located in front of the photomultiplier, and the measurement
chamber mus t rem ain sealed agains t light. T his position has been
reached if:

❑ the tube is slightly raised in the loading position
❑ the k idney-shaped holes in the measurem ent chamber cover

and the rotor are exactly aligned (see Fig. 2-7).

The spring-loaded sealing lips (see Fig. 2-6) of the tube transport
rotor will only click into the groove in the lower side of the
measurement unit cover when this position has been reached,
thus preventing any light from entering the rear measurement
chamber.

Fig. 2-7: Exact positioning of tube transport rotor

Position a

In position a, turn the rotor
anticlockwise until position b
has been reached

Position b

LB 9507 Chapter 2: Design of the Lumat

2-9

Tube detector At the front of the instrum ent is a light barr ier for detecting tubes.

This prevents an unloaded sample holder from moving into the
measurement position. In this case injection would contaminate
the inside of the measurement chamber. The test is performed
every time the <start> button is pressed during the meas urement
procedure or when activating the washing function.

Stepping motor The tube transport rotor is driven by a stepping motor which can

be stopped by applying a slight amount of pressure. T his prevents
finger injury and stops the tubes from getting damaged. If you
want to clean the upper surface of the tube transport rotor, you
can turn it manually to any position. To return the rotor to the
correct position, press the sof tkey <TURN ONCE>. This will turn
the rotor to the next position or the position after next, depending
on the current position. You can reset the pos ition m anually if you
like.

Injector head T he injector head (only on instruments with injectors) is fixed to

the measurement c hamber cover by 2 silver-coloured cross-head
bolts. It incorporates a ring-shaped raised sec tion with a sealing
ring which sits on the opening on the side of the measurement
chamber cover (Fig. 2-8).

Do not open the small cover on the injector head.

Fig. 2-8: Placing the injector head on the sample holder

Injector head

Ring-shaped raised

section with sealing ring

Measurement

chamber

Tube detector

Chapter 2: Design of the Lumat LB 9507

2-10

2.5 Reagent Storage Area

A maximum of 2 reagent bottles are stored in an eas ily acces s ible
recess on the top of Lumat. The containers are filled with the
reagents required for the applications in hand, and linked to
connectors 1 and 2 next to the storage bottles on the instrument
by KEL/F screw joints (Fig. 2-9). Inside the instrument, thes e lines
lead to the injectors, and from there to the injector head with its
two injector tips.

To clean the injector lines, fill the tank s with washing solution and
select the program function <WASH>.

Injector line T-piece Sealing ring T-piece

Hexagon bolt Injector l i ne

Fig. 2-9: Connection of the injector lines

LB 9507 Chapter 2: Design of the Lumat

2-11

2.6 Variable injectors

High-precision pumps inject the reagent into the chamber. The
user can define the injector volume. The integrated software
allows you to select the injectors as requir ed (no injec tion, inj ector
1, injector 2, or injector 1 and 2) and to define their volumes in 5
µL steps in the range of 25 µL to 300 µL. If the user enters the
injector volume not within the 5 µL step range, the system rounds
up or off. Values out of the range are adjus ted to the limits ( 25 µL
or 300 µL).

Flexible delay times can also be selected between the injections
and measurement. Injectors developed by Berthold and the
precise arrangement of the injector tips ens ure that the reagent is
mixed with the sample immediately upon injection, and that
exactly the right volume of reagent is used.

The injector pumps are located on the right hand side of the
instrument beneath the reagent bottles (Fig. 2-10), and are
covered by a metal plate.

Reagent 1 Reagent 2

Injector pump 1 Injector pump 2

Fig. 2-10: Lumat injectors

Chapter 2: Design of the Lumat LB 9507

2-12

Injector lines

The connections for the injector lines on the instrument are
defined as 1 (injector 1) and 2 (injec tor 2) . Use thes e des ignations
when programming measurement protocols.

The injector lines lead to the corresponding pumps, and from
there to the injector head. The right hand connection on the
injector head comes from injector pump 1 (see Fig. 2-11).

Fig. 2-11: The connections injector pumps / injector head

Injector pump 2

Injector pump 1

Injector head

Measurement
chamber

LB 9507 Chapter 2: Design of the Lumat

2-13

2.7 Reagent Supply

Lumat is available in different versions:

1. without injectors (with septum for manual injections)

2. with one variable injector

3. with two variable injectors.

Setting the injector volume

For instruments with one or two injectors , you enter the desired
injector or injectors together with their volumes once in the
<INSTRUMENT PARAMETERS> menu. The sof tware us es these
settings when creating protocols and perform ing measurements.
When creating protocols the settings entered in the instruments
parameter can be changed.

Setting ”0” means the respective inj ector is dis abled and does not
appear in the protocol prompts.

Injector vol umes:
25 µL - 300 µL in 5 µL steps
If the user enters the injector volume not within the 5 µL step
range, the system rounds up or off. Values out of the range are
adjusted to the limits (25 µL or 300 µL).

Example:
When entering 43 µL the system rounds up to 45 µL, when
entering 102 µL the system rounds down to 100 µL.

Wash function and selected injectors:

If the wash function is selected in the <REAGENTS> menu, only
the injector lines are filled and washed in the case in which an
injector volume > 0 has been entered in the <INSTRUMENT
PARAMETERS> menu.

This manual descr ibes the version with 2 injectors, s ince this text
covers all other options. So, if your Lumat does not include an
injector, you can skip all inform ation relating to injectors such as
selecting injectors when creating protocols, injecting during
measurement, etc.

Chapter 2: Design of the Lumat LB 9507

2-14

2.8 Memory Management

The Lumat LB 9507 memory manager has the following structure:

PC

Operating software
with or without
custom modules

CPU

Internal Memory

Data from 30 most
recent runs

Max. of 40 measure­ment protocols

Instrument
parameters

Instrument

configuration
Measurement
electronic Working memory

Fig. 2-12: Structure of the memory manager in Lumat LB 9507

Memory structure

The internal memory contains the instrument and configuration
parameters, as well as the measurem ent protocols and data fr om
the 30 most recent runs. Par t of this memory is used as a work ing
memory for measurements and evaluations.

Storage security

A maximum of 40 measurement pr otocols can be created by the
user, and these are stored in the instrument’s internal memory
independent of current and battery, as are the instrument and
configuration parameters . Therefore, these can only be lost if the
CPU develops a fault, or similar.

In the event of a power failure, the data from the 30 most recent
runs are stored in a battery-backed memory for at least 3 months.

LB 9507 Chapter 2: Design of the Lumat

2-15

Operating - software

Fig. 2-13: Data transfer: internal memory - PC

Loading operating software

The instrument’s RS232 interface enables you to load the
operating software from a PC to Lumat.
From the terminal pr ogram, loading the operating software via a
serial cable is a matter of a few mouse-clicks.
This transfer proc edure should be carried out when you receive
an update or upgrade on diskette or by email.

Procedure for loading the operating software

Use a PC to load the operating software from the diskette or email
to Lumat. The existing sof tware on the m em ory will be over written
and all measurement protocols on the memory will be deleted,
that new protocols can be stored.

❑ Switch Lumat on.
❑ Connect Lumat (host I/O board) and the PC using a serial

cable.

❑ Prepare the instrument for receiving data by entering the

service function “S4”.

Chapter 2: Design of the Lumat LB 9507

2-16

❑ Start the term inal program, which is along the new operating

software on the diskette or email, and load the new software
to Lumat. This overwrites the m emory with the new operating
software, deleting all measurement protocols. For better
instructions see text file "READ.ME", which is inc luded in the
software update package.

In all cases, the contents of the inst rumen t’s in ternal memo ry
remain unchanged (instrument and configuration
parameters, measurement modes).

LB 9507 Chapter 3: Getting Started

3-1

3. Getting Started

3.1 Instrument Setup

The place where the instrument is set up should have the
following characteristics:

- free of dust

- no strong magnetic fields

- no direct exposure to sunlight

- ambient temperature +15 - +30°C

- relative humidity 10 - 90 %, no condensation

- power supply as specified on the type label

- distance of rear panel from the wall at least 10 cm to provide
the necessary cooling

- operation area above the instrument at least 20 cm

- firm, smooth surface leaving the air filter inlet open

3.2 Power Supply

A selection of the line voltage is not necessary. The
multi range power supply will detect the line voltage
automatically , only make sure that the correct lin e fuse
is fitted.

1 Amps slow blow for 230 V
2 Amps slow blow for 115 V

at a frequency of 47 - 62 Hz.

☞☞☞☞ Use only the cable supplied with the instrument to

connect it to the mains!

3.3 Checking the Paper Supply

Check if the paper supply is sufficient; for changing paper see
section 13.1.

Chapter 3: Getting Started LB 9507

3-2

3.4 Switching on Lumat

Please watch the display:

1. After the power is switched on, the following messages

should appear:

ROTATING TO INITIALISE

When this message appears, the tube transport rotor will
perform a whole revolution. The ”ready” message then
appears with the main menu:

READY <DATE> <TIME>
MEASURE PROTOCOL -OTHERS-

If not, the microprocessor has detected an error (see Chapter

14.1).

2. If "MEMORY ERASED" is displayed together with the

"READY" message after the power is switched on, the stored
data has been lost due to a memor y defect. This will also be
indicated by a 3 second beep. You can work with the
instrument after re-entering all configuration and instrument
parameters.

LB 9507 Chapter 3: Getting Started

3-3

3.5 System Configuration Parameters

The parameters listed in this chapter are valid f or all pr otoc ols and
measurement types, and are therefore default values that are
essential for operation of the instrument (s ee Chapter 14.4). The
parameters entered rem ain stored in the lum inom eter, even if it is
switched off (except for the time and date, which remain stored for
approximately one month).

Please verify all configuration parameters when installing the
instrument.

These parameters are entered in the <SYSTEM
CONFIGURATION> menu. They describe the general mode of
operation of the LB 9507.

The entry menu is accessed as follows: in the main menu ( with
the READY display), press the k ey below <OTHERS>. Press this
key again in the next display. The next display appears, containing
the <SYSTEM CONFIGURATION> menu. Press the key below
this option to display the configuration parameters.

READY <DATE> <TIME>
MEASURE PROTOCOL -OTHERS-

<DATE> <TIME>

OPER. FUNCT. TURN ONCE -OTHERS-

<DATE> <TIME>

SYST. CONFIG. INSTR.PARAM. -OTHERS-

Fig. 3-1: Operate the softkey typed in bold face to access the

configuration parameters.

Chapter 3: Getting Started LB 9507

3-4

3.5.1 Description of the System Configuration Parameters

a) Calculating the Coefficient of Variation

<DATE> <TIME>

SYST. CONFIG. INSTR.PARAM. -OTHERS-

INSTRUMENT CONFIGURATION
CV/CVD LANGUAGE -OTHERS-

COEFFICIENT OF VARIATION CALCULATED FROM
RLU (C.V.) CONC. (CVD.)

<C.V./CVD.>: this option is available only when using the internal
thermal printer. By selecting either <RLU (C.V.)> or <CONC.
(CVD.)> you can specify here whether, in the assay mode, the
calculated coefficient of var iation should be printed out in RLU or
units of concentration.

b) Selecting the Language

INSTRUMENT CONFIGURATION
CV/CVD LANGUAGE -OTHERS-

SELECT LANGUAGE
ENGLISH GERMAN FRENCH

<LANGUAGE>: here you define the desired user language.
German, English and French are available.

c) Setting the Date and Time

INSTRUMENT CONFIGURATION
CV/CVD LANGUAGE -OTHERS-

INSTRUMENT CONFIGURATION
DATE TIME -OTHERS-

<DATE>: enter date (DDMMMYY).

LB 9507 Chapter 3: Getting Started

3-5

The date and time will continue to run for one month with the LB
9507 switched off. The built-in clock is supplied by a small
rechargeable battery, making it independent of memor y activities
in the instrument.

<TIME>: enter time and time format. You can select either 24­hours <24HR>, or <AM> and <PM>, which are displayed after you
have selected <TIME> in the second line of the display. Separate
the date and time by pressing the point key.

d) Beep

INSTRUMENT CONFIGURATION
CV/CVD LANGUAGE -OTHERS-

INSTRUMENT CONFIGURATION
DATE TIME -OTHERS-

INSTRUMENT CONFIGURATION
PRINTER READY BEEP -OTHERS-

BEEP WHEN MEASUREMENT FINISHED : OFF
OFF SHORT LONG

You can decide whether you want the luminometer to beep when
the measurement is f inished. You can select SHORT, LONG or
no (OFF) beep.

Chapter 3: Getting Started LB 9507

3-6

3.5.2 System Configuration Table

Configuration Alternatives

Language German/English/French
Coefficient of CV or CVD
variation (only required for thermal printer)
Time 0 to 24 hrs or AM/PM; entry
Date entry
Ready beeper short, long or off

LB 9507 Chapter 3: Getting Started

3-7

3.6 Instrument Parameters

3.6.1 Setting/Verifying the Instrument Parameters

The instrument parameters are those instrument settings which
are valid for all measurements. This is where the mechanical
times for the injector pumps and the instrument sensitivity are
standardised using a certain calibration factor.

For luminometers with one or two injectors installed, this is where
you define which injector(s) is/are to be activated, along with their
volumes. The activated injectors and injector volumes can be
changed here (default values) or in the parameter protocols
(protocol specified) when needed (see chapter 2.7 for volume
ranges).

The best way to check the presently defined instrument
parameters is by producing a param eter printout which is created
as follows (press the softkeys in bold face):

READY <DATE> <TIME>
MEASURE PROTOCOL -OTHERS-

<DATE> <TIME>

OPER. FUNCT. TURN ONCE -OTHERS-

<DATE> <TIME>

SYST. CONFIG. INSTR. PARAM. -OTHERS-

ENTER CALIBRATION FACTOR FOR BLACK RLU 1.00
PRINT PARAM.

ENTER CALIBRATION FACTOR FOR RED RLU 1.00
PRINT PARAM.

These values are factory set and do not usually have to be
changed. You will only have to enter them again in the event of a
memory loss in which some or all of these data are des troyed. For
your instrument, these parameters are:

Chapter 3: Getting Started LB 9507

3-8

Cal. Factor for red RLU

Cal. Factor for black RLU

Injector volume Inj. 1 (µL):

Injector volume Inj. 2 (µL):

red: measurement chamber marked with a red spot
black: measurement chamber unmarked.

Please enter these values listed in your test protocol before you
start up your instrument and compare them with the printout.
Correct the stored values only if the values in your printout and in
the test protocol do not match (see chapter 14.4).

LB 9507 Chapter 3: Getting Started

3-9

3.6.2 Changing the Instrument Parameters

The instrument parameters of the LB 9507 are edited as follows:

READY <DATE> <TIME>
MEASURE PROTOCOL -OTHERS-

<DATE> <TIME>

OPER. FUNCT. TURN ONCE -OTHERS-

<DATE> <TIME>

SYST.CONFIG. INSTR.PARAM. -OTHERS-

ENTER CALIBRATION FACTOR FOR RED RLU 1.00
PRINT PARAM.

ENTER CALIBRATION FACTOR FOR BLACK RLU 1.00
PRINT PARAM.

These values must match the RLU value of the test protocol. It
refers to the inherent sensitivity of the photomultiplier . Confir m the
value by pressing <enter>.

If a printout of the instrument parameters is required, press the
button beneath <PRINT PARAM.>.

ENTER VOLUME INJ. 1 300
PRINT PARAM. LAST DISPLAY

Enter the volume of injector 1 in µL. This display appears only for
instruments with at least one injector. If you enter “0”, injec tor 1 is
disabled for all measurements.

ENTER VOLUME INJ. 2 [µl] 200
PRINT PARAM. LAST DISPLAY

Enter the volume of injector 2 in µL. This display appears only for
instruments with 2 injectors. If you enter “0”, injector 2 is disabled.

Next, you are asked:

Chapter 3: Getting Started LB 9507

3-10

CHANGES COMPLETE?
YES NO PRINT PARAM.

Entering <YES> stores the parameters and returns the program to
the menu.
Entering <NO> returns the program to the first instrument
parameter.

LB 9507 Chapter 3: Getting Started

3-11

3.7 Reagent Supply

Depending on the requirements for the luminescence reaction, the
instrument may operate with 0, 1 or 2 inject ors. Accordingly, you
have to install one or two reagent bottles. Two empty bottles are
included with the instrument. Kit manufacturers supply their
specific activator and starter reagents in suitable bottles.

Technical data about the injector system

Injector volume: 25µL - 300µL in 5µL steps
Injection time: less than 0.6 seconds
Precision: ± 3µL for the total range;

<0.5% (300µL <0.1%)

Handling of injectors

Injector lines have to be washed before starting an assay. Fill
lines with reagents. After each experim ent the injector lines have
to be cleaned and the liquid has to be removed from the system.

Filling of the injector lines

Proceed as follows:

a) Switch on the instrument.

b) Make sure that the setting of the injectors is correct (see

<INSTRUMENT PARAMETERS>, section 3.8.1).

c) From the <OPERATOR FUNCTIONS> menu, select the

function <REAGENT> <PRIME> to fill up the supply lines
and pump(s).

Pumping back the reagents

After measurem ent, the reagents left in the injector system, can
be pumped back into the reagent bottles (see section 4.2.3,
<MANUAL UNLOAD> function in the <OPERATOR
FUNCTIONS> <REAGENT> menu).

Washing of injector lines after the measurement

After finishing measurement the injector system should be
cleaned. Injector lines are made of T eflon/T ef zel and are resistant
to a variety of different chemicals. For cleaning the lines you
should use a lye or an acid (e.g. 0.3 M NaOH or 0.3 M HCL).
Then rinse lines several tim es with H

2

0. Remove the liquid from

Chapter 3: Getting Started LB 9507

3-12

the injection lines if the instrument will not be used for a long
period of time.

LB 9507 Chapter 3: Getting Started

3-13

3.8 Safety Precautions for Operation

1. Operate the instrument in dry environments only.

2. Be careful when handling strong alkaline or acidic reagents. Be
sure to follow the reagent manufacturer’s safety instructions
(R&S-set).

3. If there is a leak in the reagent tubing system you must abort
the measurement s eries and eliminate the leak. If nec essary,
call the manufacturer’s T echnical Ser vice. If there is a leak the
reagents may flow on the instrument desk.

4. Only service technicians authorized by Berthold Technologies
are allowed to repair or modify the instrument.

LB 9507 Chapter 4: Structure and Operation of Software

4-1

4. Structure and Operation of Softw are

4.1 Operation

The software of the Lumat LB 9507 is menu-based. The main
menu comprises 5 subm enus c ontaining a wide range of progr am
functions. Commands or parameters are entered via the
respective menus.

You can select a menu and run through the program by means of
function keys and so-called softkeys. Softkeys are keys with
changing functions defined by the software and shown on the
display. The functions are selected using the corresponding
buttons beneath the display.

There are three function keys used for operating the program,
each with a fixed meaning:

<enter> to confirm entries
<delete> to delete the most recently entered text or numerical

values

<exit> to abort a command or program procedure and to

return to the main menu

The keyboard includes a complete s et of alphanumeric characters
and a spacebar.

6

exit delete last enter

1 2 3 4 5 6 7 8 9 0 .

Q W E R T Y U I O P

A S D F G H J K L -

space Z X C V B N M /

21

Fig. 4-1: Keyboard of Lumat LB 9507

5

4

3

Chapter 4: Structure and Operartion of Software LB 9507

4-2

4.2 The Six Functions of the Main Menu

You can select various functions of the main menu from the
READY screen on the Lumat LB 9507. By pressing the softkey
<OTHERS> you can switch through a loop containing the various
menu displays. If no entry follows in 10 seconds, the program
automatically returns to the READY display with the
<MEASUREMENT> and <PROTOCOL> menus.

READY <DATE> <TIME>
MEASURE PROTOCOL -OTHERS-

<DATE> <TIME>

OPER. FUNCT. TURN ONCE -OTHERS-

<DATE> <TIME>

SYST. CONFIG. INSTR. PARAM. -OTHERS-

Fig. 4-2: Main menu options

Pressing the other softkeys opens the associated menus and
leads you to the options involved. The following sections provide
an overview.

LB 9507 Chapter 4: Structure and Operation of Software

4-3

4.2.1 <MEASURE> Menu

The <MEASURE> menu offers various options for running a
measurement:

READY
MEASURE PROTOCOL -OTHERS-

SELECT TYPE OF MEASUREMENT ?
PROTOCOLS DIRECT ENTRY RATEMETER

Fig. 4-2: Measurement options

<PROTOCOLS> This calls up a stored protocol which defines

the measurement mode (raw data
measurement, LIA, Cut-of f, T UP/FTI, etc .) and
the parameters for the following m eas urement,
and starts the measurement. Depending on
the selected protocol you will be given
instructions on how to operate Lumat.

<DIRECT ENTRY>Here you can enter parameters f or a raw data

measurement and then star t the m eas urem ent
directly. The most recently entered param eter
set remains stored.

<RATEMETER> You can perform a continuous measurement

of any length you choose, and the
measurement intervals can be selected as
required. During the measurement, any
number of injections can be performed using
both injectors.

Chapter 4: Structure and Operartion of Software LB 9507

4-4

4.2.2 <PROTOCOL> Menu

In the <PROTOCOL> menu you select the protocol types for the
desired measurement mode and enter the parameters for the
measurement. T he following protocol types can be selected after
you have selected the option <CREATE> in this menu:

READY
MEASURE PROTOCOL -OTHERS-

PROTOCOL
MASTER CURVE CREATE CHANGE

06: PROTOCOL NAME

<enter>

06: PROTOCOL TYPE
RAW DATA CUTOFF -OTHERS-

06: PROTOCOL TYPE
QUANT TUP/FTI -OTHERS-

06: PROTOCOL TYPE
DL-ASSAY REPEATED -OTHERS-

Fig. 4-4: Selection of protocol types

<RAW DATA> Protocol for raw data measurements.
<CUTOFF> Protocol for qualitative measurements. Using negative and

positive standards, RLU-ranges are def ined which are marked in
the printout of results.

<QUANT.> Protocol for LIA measurements with or without master curve. To

run an LIA measurement with m as ter cur ve you only have to enter
a short protocol here. Select <MASTER CURVE> and enter the
data for the Master Curve in this m enu. A master curve can only
be attached to an existing LIA protocol without its standard series.

LB 9507 Chapter 4: Structure and Operation of Software

4-5

<TUP/FTI> This protocol type is used to calculate the patient data using a

standard.

<DL-ASSAY> Automatic measurement of the Dual-Luciferase

TM

Reporter

Assay. (Injection-measure-injection-measure)

<REPEATED> Protocol for the meas urement of long time k inetics us ing repeated

measurements of the same sample within a fixed timed interval;
with or without injection.

Chapter 4: Structure and Operartion of Software LB 9507

4-6

4.2.3 <OPERATOR FUNCTIONS> Menu

This menu includes the following user functions (for a description
see Chapter 11):

<REAGENT> Loading, refreshing and removing reagents and washing of

injectors
<PERF. TEST> Instrument and reagent test
<MANUAL DATA > Manual entry of RLU data for calculation with evaluation protocols
<RERUN> Calls up stored data for re-calculation and printout.

READY
MEASURE PROTOCOL -OTHERS-

READY
OPER. FUNCT. TURN ONCE -OTHERS-

OPERATOR FUNCTIONS:
REAGENT PERF.TEST -OTHERS-

OPERATOR FUNCTIONS:
MANUAL DATA RERUN -OTHERS-

Fig. 4-5: Operator functions

The function <REAGENT> is only provided for instruments with at

least one injector installed.

LB 9507 Chapter 4: Structure and Operation of Software

4-7

4.2.4 <SYSTEM CONFIGURATION> Menu

Here you define the instrument configuration parameters:
<CV/CVD> Specify here whether the coefficient of variation is to be s pecified

in RLU (=C.V.) or in concentration units (CVD) on the thermal

printout.
<LANGUAGE> Set the language which you want to use in the integrated

software’s user interface (German/English/French).
<DATE> Enter the date.
<TIME> Enter the time.
<BEEP> Switching on and off the internal beep when the m easurement is

finished

READY
MEASURE PROTOCOL -OTHERS-

<DATE> <TIME>

OPER. FUNCT. TURN ONCE -OTHERS-

<DATE> <TIME>

CONFIG. INSTR. PARAMS -OTHERS-

INSTRUMENT CONFIGURATION
CV/CVD LANGUAGE -OTHERS-

INSTRUMENT CONFIGURATION
DATE TIME -OTHERS-

INSTRUMENT CONFIGURATION
READY BEEP -OTHERS-

Fig. 4-6: Instrument configuration

Chapter 4: Structure and Operartion of Software LB 9507

4-8

4.2.5 <INSTRUMENT PARAMETER> Menu

Enter and verify the instrument parameters, e.g. the volume of the

injectors used or the calibration factors (s ee section 3.8). Fig. 4-6

shows you how to select this menu.

4.2.6 <TURN ONCE> function

When you select this func tion, the measurem ent cham ber rotates

through 180°. This allows you to move a sample tube into the

measurement position or out of the measurem ent position while

not actually measuring (useful when cleaning).

READY
MEASURE PROTOCOL -OTHERS-

<DATE> <TIME>

OPER. FUNCT. TURN ONCE -OTHERS-

Fig. 4-7: Selecting the <TURN ONCE> function

4.2.7 Service functions

The program contains the following service functions; these are

only accessible to Berthold Technologies’ customer service

engineers.

<INJECT. TEST> Injector test.

<PM TEST> Tests the photomultiplier.

<ROTOR TEST> Tests the measurement chamber rotor.

<MONITOR> Loads new operating software.

LB 9507 Chapter 4: Structure and Operation of Software

4-9

4.3 General Procedure

The following flow chart provides an overview of the essential

functions of Lumat, together with the way in which they are

interrelated. More detailed information is available in the c hapters

listed.

1. Getting Started

The instrument parameters are defined and the instrument is

configured during the commissioning procedure.

2. Creating protocols for routine measurements

Up to 40 protocols can be stored in the <PROTOCOL> menu for

routine measurements. Four different types of protocols are

available.

3. Measurements

The <MEASURE> menu includes the following options:

● Measurements based on stored protocols

● Raw data measurement with direct entry of the parameters

● Kinetics measurement

4. Operator Functions

The <OPERATOR FUNCTIONS> menu provides the following

options:

● Data evaluation with manual data entry

● Evaluation of stored data

● Performing a system test

● Washing of the injector system

● Re-pumping of the reagents

Chapter 4: Structure and Operartion of Software LB 9507

4-10

Fig. 4-8: Illustration of the general procedure

1. Getting started
Chapter 3

Connect instrument

Setting the instrument parameters

Injector volume, calibration factors etc.

Instrument configuration

Language, date, time, printer

Calculating the coefficient of variation

Chap. 5 Chap. 6 Chap. 8 Chap. 7 Chap. 7 Chap. 9 Chap. 5 Chap. 10

2. Protocol
setu

p

Raw
data

Cut-off UP/FTI Quant. Master

curve

DLR

A

ssa

y

3. Measurements

Raw
data

meas.

Cut-off

meas.

UP/FTI

meas.

Quant.

meas.

DLR

Assay

Raw data

direct
meas.

Kinetic

meas.

4. Operator
functions
Chapter 11

Evaluation

of stored data

Reagents Performance

Test

Data evaluation

with manual data

input

Prime reagents

LB 9507 Chapter 5: Raw Data Measurements

5-1

5. Raw Data Measurements

5.1 Introduction

In the Raw data measurement mode the quantity of light

measured over a user-defined measuring time is measured in

RLUs for each sample. You have the option of printing out a high

resolution (up to 2000 data point) curve and/or up to 50 data

points with measurement time for each sample, which enables

you to analyze the kinetic behaviour of the light emission of a

sample.
Replicate groups can also be defined for which the aver age value

and the variation coefficient are calculated and printed out.

This measurement mode, which allows up to 2 injections per

sample, is used mainly for ATP measurements and for

measurement of luminescence reporter gene assays.

There are two ways of starting raw data measur ement (see Fig.

5-1):

1. Create and save protocols for routine measurements in the
<PROTOCOL> menu (max. 40). W ith each of these protocols
you can later start as many measurements as you want in the
<MEASURE> menu.

2. Running a raw data direct measurement.
Select the option < MEASURE > from the <DIRECT ENT RY>

menu to enter the measurement parameters. Then start the
measurement.

In both cases the parameters you are asked to enter are the
same.

The displays are numbered consecutively from R1... R34 for
clarity.

The following illustration shows a flow chart of the protocol setup
procedure and measurement start for routine and direct
measurements.

Chapter 5: Raw Data Measurements LB 9507

5-2

Direct measurements

R1

READY
MEASURE PROTOCOL -OTHERS-

R2

PROTOCOL
MASTER CURVE CREATE CHANGE

R3 R2a

R2b

R4 R2b

R2c

R5

06: OPERATOR ID.

(…)

R19

06: INPUT ALL CORRECT? YES
YES NO

R1

READY
MEASURE PROTOCOL -OTHERS-

R21

SELECT TYPE OF MEASUREMENT ?
PROTOCOLS DIRECT ENTRY RATEMETER

R22 R6

R19

Fig. 5-1: Flow chart showing protocol setup and measurement

Create protocol

Change protocol

06: PROTOCOL NAME

06: PROTOCOL NAME
RAW DATA CUTOFF -OTHERS-

06: PROTOCOL NUMBER 6
PRINT LIST

PROTOCOL 6
CHANGE OTHERPROT.PRINT PROT.

DELETE PROTOCOL?
NO YES

Routine measurements

ENTER PROTOCOL NUMBER 6

06: INPUT ALL CORRECT ? YES
YES NO

06: ENTER INJECTOR VOLUME 1 [µL]
PRINT LIST LAST DISPLAY

Start of measurement

LB 9507 Chapter 5: Raw Data Measurements

5-3

5.2 Creating Raw Data Protocols

For routine measurements, start by entering the parameters for
the measurement procedure and the evaluation strategy in a
measurement protocol in the <PROTOCOL> menu, and then
save them. Later, you can run a measurem ent simply by calling
up one of these stored protocols in the <MEASURE> menu and
pressing the <START> button.

You can save up to 40 different measurement protocols which
include all information such as injector control, definition of
replicates, measuring time etc., as well as information on data
evaluation. Each new protocol is given a number which is
dependent upon the time the protocol was set up and independent
of the protocol type (raw data, cut-off...).

To create a protocol you have to run through displays R1 to R18.
Most displays include the option <LAST DISPLAY> which takes
you back to the previous display.

Chapter 5: Raw Data Measurements LB 9507

5-4

R1

READY
MEASURE PROTOCOL -OTHERS-

Select the <PROTOCOL> menu by pressing the button located
directly below. The following display appears:

R2

PROTOCOL
MASTER CURVE CREATE CHANGE

The <MASTER CURVE> option is not relevant to raw data
measurements.
Select <CHANGE> to open an existing protocol by entering its
protocol number. You can then edit it.
To create a new protocol, select <CREATE>. You will see this:

R3

06: PROTOCOL NAME ATP-10SEC-1INJ

The next free number is displayed for the new protocol (in our
example “06:”). Enter a name for this protocol using the
alphanumeric keyboard. You should use a name which specifies
the protocol type and other important features, so that it is easy to
identify later. For example “ATP-10SEC-1INJ”.
Confirm your entry with <enter>.

R4

06: PROTOCOL TYPE
RAW DATA CUTOFF -OTHERS-

Select <RAW DATA>. Now you have to enter the parameters
which are the same for all protocol types:

R5

06: OPERATOR ID.

Enter the user name and press <enter>.

LB 9507 Chapter 5: Raw Data Measurements

5-5

R6

06: ENTER INJECTOR VOLUME 1 [µl]

LAST DISPLAY

Changing or confirmation of the displayed injector volume.
Displayed are the standard settings for injector 1 entered in the
<INSTRUMENT PARAMETERS> menu. These settings can be
changed for this specific protocol.
Setting "0" means the respective injector is disabled.
Range: 25 µL - 300 µL in 5 µL steps.

If the user enters the injector volume not within the 5 µL step
range, the system rounds up or down. Values out of the range are
adjusted to the limits (25 µL or 300 µL).
Example: W hen entering 43 µL the system rounds up to 45 µL,
when entering 102 µL the system rounds down to 100 µL.

Press <enter> to use the response displayed in the upper-right
corner of the LCD.

R7

06: ENTER INJECTOR VOLUME 2 [µl]

LAST DISPLAY

Changing or confirmation of the displayed injector volume.
Displayed are the standard settings for injector 2 entered in the
<INSTRUMENT PARAMETERS> menu. These settings can be
changed for this specific protocol.
Setting "0" means the respective injector is disabled.
<LAST DISPLAY> takes you back to the previous display.
Setting "0" takes you to display R10. Setting >0 takes you to
display R8. Press <enter> to use the response displayed in the
upper-right corner of the LCD.

R8

06: SEQUENCE OF INJECTIONS 1->2
1->2 2->1 LAST DISPLAY

If you have selected 2 injectors, you have to define the sequence
of the injections here: injector 1 first and then injector 2 or vice
versa. Make your selection by pressing the button below the
option you want. The following display appears:

Chapter 5: Raw Data Measurements LB 9507

5-6

R9

06: DELAY TIME INJ1/INJ2 1.2
(1.2 - 300.0) LAST DISPLAY

Enter the desired delay in seconds (1.2 to 300 sec) between both
injections and confirm with <enter>.
Only if you have selected one or two injectors:

R10

06: MEASURE BACKGROUND NO
YES NO LAST DISPLAY

Select here if the background r ate is to be measured prior to the
injections and the actual measurement of each tube.
If you select <NO>, display R14.
<YES> takes you to the next display:

R11

06: MEASURING TIME BKG [s] 0.5
(0.1 - 200.0) LAST DISPLAY

Enter between 0.1 and 200.0 seconds for the background
measurement and pres s <enter>. Reasonable times start at half
a second, allowing statistical variations to be compensated for.
Otherwise, the background values may be too high or too low
which either distorts the result of the m easurem ent or leads to an
exceeding of the threshold.

Remember that the background effect is measured in order to
obtain an alarm threshold for background rates that ar e too high,
as with phosphorescence effects for example.

R12

06: AUTOMATIC BKG SUBTRACTION NO
YES NO LAST DISPLAY

Here you can select whether the background effect is to be
subtracted automatically from the measured results.

Remember that the statis tic al signif ic ance of the meas ured values
is dependent upon the measuring time.

LB 9507 Chapter 5: Raw Data Measurements

5-7

R13

06: MAX. BACKGROUND [RLU/s] 50
(0 = NONE) LAST DISPLAY

Here can you define the limit value for the background. If this
threshold is exceeded, you will be alerted accordingly on the
display and the printout (BKG).
Entering “0” means “no background threshold“.
As soon as you confirm the entry with <enter> you move to the
next display.

R14

06: DELAY LAST INJ./MEAS. [s] 1.0
(0.0 - 300 s) LAST DISPLAY

Enter the delay between the last injection and the start of the
measurement in the range from 0.0 to 300 seconds and press
<enter>.

R15

06: MEASURING TIME [s] 2.0
(0.1 - 200.0) LAST DISPLAY

Enter the desired measuring time (0.1 to 200.0 seconds) for each
tube and press <enter>.

R16

06: NUMBER OF REPLICATES 2
(1 - 10) LAST DISPLAY

Enter the number of replicates and pres s <enter>. You can define
a maximum of 10 sample tubes as replicates whose measured
values are averaged. “1” means 1 tube, i.e. no replicates.

R17

06:KINETICS PRINTOUT DATA YES
YES NO LAST DISPLAY

The measurement results can be printed in the form of kinetics
data on the built-in thermal printer.

Chapter 5: Raw Data Measurements LB 9507

5-8

R18

06:KINETICS PRINTOUT CURVE YES
YES NO LAST DISPLAY

The measurement results can be printed in the form of kinetics
curve on the built-in thermal printer.

R19

06: INPUT ALL CORRECT ? YES
YES NO LAST DISPLAY

If you select <YES> the entered parameters are stored and the
program returns to the main menu (R1).
<NO> takes you back to the 1st display of this protocol (R5).

Note:

The protocol is not stored if you exit this protocol sequence via
<exit>!

LB 9507 Chapter 5: Raw Data Measurements

5-9

5.3 Editing/Deleting Protocols

R1

READY
MEASURE PROTOCOL -OTHERS-

Select the <PROTOCOL> menu by pressing the button located
directly below. Then follows:

R2

PROTOCOL
MASTER CURVE CREATE CHANGE

Select <CHANGE>.

R2a

ENTER PROTOCOL NO. 6
PRINT LIST

You can print the list of stored protocols by selecting <PRINT
LIST>. Enter the required protocol number and press <enter>.

R2b

PROTOCOL # 6: ATP-10SEC-1INJ
CHANGE OTHER PROT. PRINT PROT.

Select <PRINT PROT.> to print out the selected protocol.
<OTHER PROT.> takes you back to the previous display and lets
you enter a new protocol number.
< CHANGE> takes you to the next display:

R2c

DELETE PROTOCOL NO. 6:
NO YES

To delete this protocol, select <YES>.
To edit the protocol, select <NO>. If you do this, the first entry of
this protocol is displayed, i.e. the user name in display R5:

Chapter 5: Raw Data Measurements LB 9507

5-10

R5

06: OPERATOR ID

This and all the following entries can be overwritten. T he following
input sequence is the same as the one described in the last
section starting with display R6. If you want to leave certain
parameters as they are, just press <enter>.

LB 9507 Chapter 5: Raw Data Measurements

5-11

5.4 Performing Raw Data Measurements

There are two ways of performing a raw data measurement:

1. You can use a protocol which has already been created and

stored. To do this, select the option <PROTOCOLS> in the
<MEASURE> menu.

2. Alternatively, enter the param eters for the m easurement you

want to run. To do this, select the option <DIRECT ENTRY>
in the <MEASURE> menu and enter the measurement
parameters. These are the param eters listed in displays R5
to R19. Then start the measurement.

Measurement with
stored protocol

R1

READY
MEASURE PROTOCOL -OTHERS-

Select <MEASURE>.

R21

SELECT TYPE OF MEASUREMENT ?
PROTOCOLS DIRECT ENTRY RATEMETER

Select <PROTOCOLS> to open a stored protocol.
Select <DIRECT ENTRY> to activate the input sequence for the
measurement parameters of a direct measurement.

R22

ENTER PROTOCOL NO. 6
PRINT LIST

Enter the number of the protocol required and press <enter>.
You can print out the list of stored protocols including num ber and
name by pressing the button below <PRINT LIST>.

Chapter 5: Raw Data Measurements LB 9507

5-12

R23

06: PROTOCOL ATP-10SEC-1INJ ?
YES NO

You are prompted to confirm that this is the required pr otocol. If
you select <NO> the program returns to display R22. <YES>
leads to:

R24

COMMENT

You can enter a comment on the following measurement using
the alphanumeric keyboard. Confirm it with <enter>. If you do not
want to enter a comment, just press <enter>.

R28a

SAMPLE 1 TUBE # 1
INSERT TUBE...

Load the tube containing the first sample as defined in the
protocol.

R29

SAMPLE 1 TUBE # 1

START

Press the button beneath the <START> function or the <start>
button to the right of the rotor. Both buttons do the sam e thing.
The tube will move back into the measurem ent cham ber, and the
programmed measurement procedure will begin:

R30

BACKGROUND MEASURING...

Background measur ement is performed, assum ing you specified
it in the protocol.

LB 9507 Chapter 5: Raw Data Measurements

5-13

R31

BACKGROUND TOO HIGH !

This display only appears when the background threshold you
have defined in the protocol is exceeded. If this happens the
printout of measurement values is flagged BKG.

The steps which the instrum ent goes through are displayed and
the measurement param eters f rom the active pr otocol are printed
out at the same time.

R32

INJECTING ...

R33

MEASURING...

R34

SAMPLE-RLU 133

REMOVE TUBE

Once measurem ent is complete, the first line shows the r esult of
the measurement in RLUs, the second line (if defined in the
protocol) shows the result of the background measurement. At the
same time the m easurement parameters defined in the protocol
are printed out as a protocol header.
The measured values are then printed:

Measured values output:

No kinetics curve, no replicates selected:

One measured value per sample (RLU)

No kinetics curve, but replicates:

One measured value per sample (RLU)
Average for replicate group and coefficient of variation

Chapter 5: Raw Data Measurements LB 9507

5-14

Kinetics curve (without data) selected without replicates:

Up to 2000 measurement points per sample displayed as a
curve (according to the measurement time)
Peak maximum and total integral of each sample

Kinetics curve (with data) with replicates:

20 to 50 measurement points per sample
Up to 2000 measurement points per sample displayed as a
curve
Peak maximum and total integral of each sample
Average for replicate group and coefficient of variation

When you remove the sa mple tube, the measured value display
disappears and the following is requested:

R36

SAMPLE 2 TUBE # 2
INSERT TUBE ...

Load the next sample tube.

R37

SAMPLE 2 TUBE # 2

START

Press the <Start> button. The 2nd tube is measured. If it is a
replicate, “SAMPLE 1” will remain on the display until all tubes of
this group have been measured, while the “TUBE #” is
incremented continuously.

After pressing the <Start> button, the procedur e is the same as
described for steps R29 to R34.

LB 9507 Chapter 6: CUT-OFF-Measurements

6-1

6. CUT-OFF Measurements

6.1 Introduction

The aim of CUT -OFF measurem ents is to divide patient sam ples
into 3 qualitative classes separated by 2 CUT-OFF thresholds
(LOW and HIGH CUT-OFF) using fixed parameters and controls.

The samples are marked as follows:

RLU value below LOW CUT-OFF ”NEG”
RLU value between LOW and HIGH CUT-OFF ”+/-”
RLU value above HIGH CUT-OFF “POS”

Both CUT-OFF thresholds are calculated as follows:

LOW CUT-OFF = A

L

+ BL·(neg. std) + CL (1st pos. ctrl)

HIGH CUT-OFF = A

H

+ BH·(neg. std) + CH (1st pos. ctrl)

where the following are defined in the protocol:

A

L

= LOW CUT-OFF offset (RLU) Display no. C22

A

H

= HIGH CUT-OFF offset (RLU) C25

B

L

= LOW CUT-OFF FACTOR
C23

influence of the neg. control on LOW CUT-OFF

B

H

= HIGH CUT-OFF FACTOR C26

influence of the neg. control on HIGH CUT-OFF

C

L

= LOW CUT-OFF LPOS. FACTOR C24

influence of the pos. control on LOW CUT-OFF

C

H

= HIGH CUT-OFF LPOS. FACTOR C27

influence of the pos. control on HIGH CUT-OFF

This program can be flexibly used with these parameters.

Example 1 Setting the number of controls to zero results in the CUT-OFF

thresholds being defined only from the OFFSET values.

Example 2 Setting the number of the pos. controls to zero res ults in the CUT-

OFF threshold being defined from the neg. control and - when
entered - from the OFFSET values.

Example 3 Setting B

H

and CL to zero results in the CUT-OFF thresholds being

defined only from the neg. resp. pos. controls and - when entered

- from the OFFSET values.

Chapter 6: CUT-OFF-Measurements LB 9507

6-2

Since negative controls often represent an important quality
criterion, limits can be s et in the protocol outside which a flag is
printed.
(LOW/HIGH RLU FLAG NEG. STD.)

Also, with replicate measurem ents, the values may be flagged if
their variations are too high.
(% CV FLAG FOR NEG./POS. CTRLS./SAMPLES)

These options can each be turned off by entering zero.

The displays are numbered consecutively C1, C2 ...CX for clarity.

The illustration on the next page shows a flow chart from creating
protocols to starting a measurement.

LB 9507 Chapter 6: CUT-OFF-Measurements

6-3

C1

READY
MEASURE PROTOCOL -OTHERS-

C2

PROTOCOL
MASTER CURVE CREATE CHANGE

C3 C2a

C2b

C4 R2b

C2c

C5

07: OPERATOR ID.

(…)

C33

07: INPUT ALL CORRECT? YES
YES NO

C1

READY
MEASURE PROTOCOL -OTHERS-

C41

SELECT TYPE OF MEASUREMENT ?
PROTOCOLS DIRECT ENTRY RATEMETER

C42

ENTER PROTOCOL NO. 7
PRINT LIST

Fig. 6-1: Flow chart showing protocol setup and measurement

Create protocol

Change protocol

07: PROTOCOL NAME

07: PROTOCOL NAME
RAW DATA CUTOFF -OTHERS-

07: PROTOCOL NUMBER 7
PRINT LIST

PROTOCOL 7
CHANGE OTHERPROT.PRINT PROT.

DELETE PROTOCOL?
NO YES

Measurements

Start of measurement

Chapter 6: CUT-OFF-Measurements LB 9507

6-4

6.2 Creating CUT-OFF Protocols

Start by entering the parameters for the measur ement procedure
and the evaluation method in a measurement protocol in the
<PROTOCOL> menu, and then save them. Later, you can run a
measurement simply by calling up one of these stored protocols in
the <MEASURE> menu and pressing the <START> button.

You can save up to 40 different measurement protocols which
include all information such as injector control, definition of
replicates, measuring time etc., as well as information on data
evaluation. Each new protocol is given a number which is
dependent upon the time the protocol was set up but not on the
protocol type.

To create a protocol you have to run through displays C1 to C32.

LB 9507 Chapter 6: CUT-OFF-Measurements

6-5

C1

READY
MEASURE PROTOCOL -OTHERS-

Select the <PROTOCOL> menu by pressing the respective
button. Then follows:

C2

PROTOCOL
MASTER CURVE CREATE CHANGE

The <MASTER CURVE> is not relevant to CUT-OFF
measurements.
If you press the button below <CHANGE> you can open an
existing protocol by entering its protocol number, and then edit it.
To create a new protocol, press the button below <CREATE>.
The following display appears:

C3

07: PROTOCOL NAME CUT-10SEC-1INJ

The next free number is displayed for the new protocol (in this
example “07:”). Enter a name for this protocol using the
alphanumeric keyboard. You should use a name which specifies
the protocol type and other important features, so that it is easy to
identify later. For example “CUT-10SEC-1INJ”.
Confirm your entry with <enter>.

C4

07: PROTOCOL TYPE
RAW DATA CUTOFF - OTHERS -

Select <CUTOFF>. Now you have to enter the parameters which
are the same for all protocol types:

C5

07: OPERATOR ID

Enter the user name and press <enter>.

Chapter 6: CUT-OFF-Measurements LB 9507

6-6

If you have no injectors selected (see 3.8.2) the display C15
follows. Otherwise C6 follows.

C6

07: ENTER INJECTOR VOLUME 1 [µl]

LAST DISPLAY

Changing or confirmation of the displayed injector volume.
Displayed are the standard settings for injector 1 entered in the
<INSTRUMENT PARAMETERS> menu. These settings can be
changed for this specific protocol.
Setting "0" means the respective injector is disabled.
Range: 25 µL - 300 µL in 5 µL steps.
If the user enters the injector volume not within the 5 µL step
range, the system rounds up or down. Values out of the range are
adjusted to the limits (25 µL or 300 µL).
Example: W hen entering 43 µL the system rounds up to 45 µL,
when entering 102 µL the system rounds down to 100 µL.
Press <enter> to use the response displayed in the upper-right
corner of the LCD.

C7

07: ENTER INJECTOR VOLUME 2 [µl]

LAST DISPLAY

Changing or confirmation of the displayed injector volume.
Displayed are the standard settings for injector 2 entered in the
<INSTRUMENT PARAMETERS> menu. These settings can be
changed for this specific protocol.
Setting "0" means the respective injector is disabled.
<LAST DISPLAY> takes you back to the previous display.
Setting "0" takes you to display C10.
Setting >0 takes you to display C8.
Press <enter> to use the response displayed in the upper-right
corner of the LCD.

C8

07: SEQUENCE OF INJECTIONS 1->2
1->2 2->1 LAST DISPLAY

If you have selected 2 injectors, you have to define the sequence
of the injections here: injector 1 first and then injector 2 or vice
versa. Make your selection by pressing the button below the

LB 9507 Chapter 6: CUT-OFF-Measurements

6-7

option you want. Press <enter> to use the response displayed in
the upper-right corner of the LCD. The following display is shown:

C9

07: DELAY TIME INJ 1/INJ 2 1.2
(0.7 - 300.0) LAST DISPLAY

Enter the desired delay in seconds between both injections and
confirm with <enter>.

C10

07: MEASURE BACKGROUND NO
YES NO LAST DISPLAY

Select here whether the background rate is to be measured prior
to the injections.
If you select <NO>, display C14 appears.
<YES> takes you to the next display:

C11

07: MEASURING TIME BKG [s] 0.5
(0.1 - 200.0) LAST DISPLAY

Enter between 0.1 and 200.0 seconds for the background
measurement and pres s <enter>. Reasonable times start at half
a second, allowing statistical variations to be compensated for.
Otherwise, the background values may be too high or too low
which either distorts the result of the m easurem ent or leads to an
exceeding of the threshold. Remem ber that the back gr ound eff ect
is measured in order to obtain an alarm threshold f or back ground
rates that are too high, as with phosphorescence for example.

C12

07: AUTOMATIC BKG SUBTRACTION NO
YES NO LAST DISPLAY

Here you can select whether the background effect is to be
subtracted automatically from the measured results.

Chapter 6: CUT-OFF-Measurements LB 9507

6-8

C13

07: MAX. BACKGROUND [RLU/s] 50
(0 = NONE) LAST DISPLAY

Here can you define the limit value for the background. If this
threshold is exceeded, a direction is displayed and printed out.
Entering “0” means “no background threshold“. As soon as you
confirm the entry with <enter> you move to the next display.

C14

07: DELAY LAST INJ./MEAS. [s] 1.0
(0.0 - 300) LAST DISPLAY

Enter the delay between the last injection and the start of the
measurement in a range from 0.0 to 300 seconds and press
<enter>.

C15

07: MEASURING TIME [s] 2.0
(0.1 - 200.0) LAST DISPLAY

Enter the desired measuring time (0.1 to 200.0 seconds) for each
tube and press <enter>.

C16

07: REPL. OF NEG. CTRL. (0-10) 3

LAST DISPLAY

Enter the number of replicates of the negative control and press
<enter>. You can define a maximum of 10 replicates whose
measured values are averaged.

C17

07: NUMBER OF POS. CTRL (0-3) 2

LAST DISPLAY

Enter the number of positive controls (0 to 3 allowed). If you enter
0, the displays C18 and C19 are skipped.

LB 9507 Chapter 6: CUT-OFF-Measurements

6-9

C18

07: REPL. OF POS. CTRL.1 (1-10) 2

LAST DISPLAY

Enter the number of replicates of the first positive control (up to 10
allowed).

C19

07: REPL. OF POS. CTRL.2 (1-10) 2

LAST DISPLAY

Enter the number of replicates of the second positive control (up
to 10 allowed).

C20

07: REPL. OF SAMPLES (1-10) 2

LAST DISPLAY

Enter the replicate number for all patient samples (1-10).

C21

07: LOW CUTOFF OFFSET 120

LAST DISPLAY

C22

07: LOW CUTOFF FACTOR 1.0

LAST DISPLAY

C23

07: LOW CUTOFF LPOS FACT. 0.0

LAST DISPLAY

These values are used to calculate the low CUT-OFF values:
Low Cut-off = Offset + m ean value (NEG) * Low Cut-of f factor +
mean value (1st POS.CTRL.) * LPOS. FACTOR (see section 6.1).

C24

07: HIGH CUTOFF OFFSET 120

LAST DISPLAY

Chapter 6: CUT-OFF-Measurements LB 9507

6-10

C25

07: HIGH CUTOFF FACTOR 0

LAST DISPLAY

C26

07: HIGH CUTOFF LPOS FACT. 1

LAST DISPLAY

The formula for the HIGH CUT-OFF threshold is similar to this
one.

C27

07: %CV FLAG FOR NEG. CTRL. 15

LAST DISPLAY

The negative control mean value is labelled “ HICV” in the result
printout if its coefficient of variation is higher than the value
entered here.

C28

07: %CV FLAG FOR POS. CTRL. 20

LAST DISPLAY

Positive standard mean values are labelled “HICV” if their
coefficients of variation are higher than the value entered here.

C29

07: %CV FLAG FOR SAMPLES

LAST DISPLAY

If this CV limit is exc eeded by patient samples, a flag appears in
the printout.

C30

07: LOW RLU FLAG NEG. CTRL. 200

LAST DISPLAY

LB 9507 Chapter 6: CUT-OFF-Measurements

6-11

C31

07: HIGH RLU FLAG NEG. CTRL. 600

LAST DISPLAY

Lower and upper limit for marking negative controls: a FLAG
appears in the printout when the value is not within the specified
value range.
“LOW” if measured value < LOW RLU FLAG
“HIGH” if measured value > HIGH RLU FLAG

C32

07: INPUT ALL CORRECT ? YES
YES NO LAST DISPLAY

This display appears at the end of the input sequence. If you
press the button below <YES> the entered parameters are stored
and the program returns to the m ain menu (C1) . <NO> takes you
back to the 1st display of this protocol (C5).

Note:

The protocol is not stored if you exit this protocol sequence via
<exit>!

Chapter 6: CUT-OFF-Measurements LB 9507

6-12

6.3 Editing/Deleting Protocols

C1

READY <DATE> <TIME>
MEASURE PROTOCOL -OTHERS -

Select the <PROTOCOL> menu by pressing the respective
button. Then follows:

C2

PROTOCOL
MASTER CURVE CREATE CHANGE

Select <CHANGE>.

C2a

ENTER PROTOCOL NO. 7
PRINT LIST

The list of stored protocols can be printed by selecting <PRINT
LIST>. Enter the required protocol number and press <enter>.

C2b

PROTOCOL # 7: CUT-10SEC-1INJ
CHANGE PROT. OTHER PROT. PRINT PROT.

Select <PRINT PROT.> to print out the selected protocol.
<OTHER PROT.> takes you back to the previous display and lets
you enter a new protocol number.
<CHANGE PROT.> takes you to the next display:

C2c

DELETE PROTOCOL # 7:
NO YES

To delete this protocol, select <YES>.
To edit the protocol, select <NO>. If you do this, the first entry of
this protocol is displayed, i.e. user name in display C5:

LB 9507 Chapter 6: CUT-OFF-Measurements

6-13

C5

07: OPERATOR ID

This and all following entries can be overwritten. The following
input sequence is the same as the one described in the last
section starting with display C6.

Chapter 6: CUT-OFF-Measurements LB 9507

6-14

6.4 Running Cut-off Measurements

C1

READY
MEASURE PROTOCOL -OTHERS-

Select <MEASURE>.

C41

SELECT TYPE OF MEASUREMENT ?
PROTOCOLS DIRECT ENTRY RATEMETER

Select <PROTOCOLS> to open a stored protocol.

C42

ENTER PROTOCOL NO. 7
PRINT LIST

The list of stored protocols including number and name can be
printed by pressing the button below <PRINT LIST>.
Enter the number of the desired protocol and press <enter>.

C43

07: PROTOCOL CUT-10SEC-1INJ O.K. ?
YES NO

You are prompted to confirm that this is really the protocol you
want. If you select <NO> the program returns to display C42.
<YES> leads to:

C44

COMMENT

You can enter a comment on the following measurement using
the alphanumeric keyboard. Confirm it with <enter>. If you do not
want to enter a comment, just press <enter>.

LB 9507 Chapter 6: CUT-OFF-Measurements

6-15

C48

REMOVE TUBE...

Remove the tube from the sample holder if there is one. This
display appears if there is a tube in the sample holder. So, if a
tube already is in the correct position before you start
measurement, you will have to lift the tube briefly. You will then
see:

C48a

NEG. CTRL REPL.#1TUBE#1
INSERT TUBE ...

Load the tube containing the first negative control (as defined in
the protocol).

C49

NEG. CTRL REPL.#1TUBE#1

START

Press the <start> button. The tube will move back into the
measurement chamber, and the programmed measurement
procedure will begin:

C50

BACKGROUND MEASURING...
REMOVE TUBE...

A background measurement is performed, provided you have
defined one in the protocol.

C51

BACKGROUND TOO HIGH !

This display only appears when the background threshold you
have defined in the protocol is exceeded. If this happens the
printout of measurem ent values is flagged BKG.The s teps which
the instruments goes through are dis played and the measurement

Chapter 6: CUT-OFF-Measurements LB 9507

6-16

parameters from the active protocol are printed out at the same
time.

C51

INJECTING ...

C52

MEASURING ...

C53

SAMPLE-RLU 133 REMOVE
BACKGR. (RLU/S) 30.0 TUBE

Once measurem ent is complete, the first line shows the r esult of
the measurement in RLUs, the second line (if defined in the
protocol) shows the result of the background measurement.
The results are printed in 4 columns:

- Measured RLU; for replicates this represents the mean of the
replicate group involved

- Coefficient of variation of the replicate group

-Flag

- Result: indication of the range

The results are classified ac cording to negative controls, positive
controls and samples.

After removing the sample tube, the measured value display is
cleared. You are prompted to:

C54

NEG. CTRL. REPL. # 2 TUBE # 2
INSERT TUBE ...

Load the next tube containing the sample def ined on the firs t line
(in this case the 2nd negative control). The start request will then
appear on the display:

LB 9507 Chapter 6: CUT-OFF-Measurements

6-17

C55

NEG. CTRL. REPL. # 2 TUBE # 2

START

Start the measurement by pressing the <start> button.
In accordance with the selected protocol, you are asked for the
defined number of replicates for negative and then positive
controls. Once a replicate group has been meas ured, the mean
value and the coefficient of variation for the group are given.

C56

ENTER REPL. NO. TO DISCARD
NONE

If you want to delete a measured replicate, enter the num ber of
the respective control that is indicated on the result printout.
Otherwise press the button below <NONE>.

The printout shows when a replicate has been deleted. The
deleted replicate is not removed from the memory. When you
load the measured data again (<OPERATOR FUNCTIONS:
DATA EVALUATION: STORED DATA>, see chapter 11.4), the
original measurement data will be used.

C57

CHANGE NEG. CTRL ?
YES NO

<YES>: Here you can edit the mean value. A correc tion is noted
on the printout. The change is not stored to the mem ory, so that
the original mean value will be displayed when calling the stored
measurement data (<OPERATOR FUNCTIONS: DATA
EVALUATION: STORED DATA>, see chapter 11.4).

<NO>: Continue the measurement.

Following mean value calculation and a possible correction, the

evaluation of the controls is output to the printer.

Then, the patient samples are requested and m ean value (RLU),
variation coefficient, flag and range are printed out for each
sample and each replicate group.

Chapter 6: CUT-OFF-Measurements LB 9507

6-18

LB 9507 Chapter 7: Quantitative Measurements (LIA...)

7-1

7. Quantitative Measurements (LIA...)

7.1 Introduction

This type of measurement is used for reagent kit specific
measurements to quantify analyte concentrations (LIA, ILMA,
etc.). Three measurement strategies are available:

1. Measurement with complete standard series

Known standard concentrations are measured before the
unknown samples. After transformation of the calibration curve
(either log-log or logit-log) interpolations between standards and
unknown samples take place. The curve f it is based on a cubic
spline function and is excellently suited to LIAs and ILMAs. If
necessary, the curve can be smoothed either automatically or
upon the user’s instruction.

2. Measurement with master curve

This measurement type uses 2 calibrators instead of standard
concentrations, which are provided as sam ples by the assay kit
manufacturer. Once this protocol has been c reated, the rem aining
value pairs (RLU/conc) of a standard curve are entered in the
submenu <MASTER CURVE> and are used as a logit-log
transformed master curve to analyse the samples. The value
pairs are included with the respective batch of reagent kits.

The displays are numbered consecutively L1, L2 ...LX for clarity.

Fig. 7-1 is a flow chart of the protocol setup procedure, entry of
the master curve and start of the measurement.

Chapter 7: Quantitative Measurements (LIA...) LB 9507

7-2

Create Protocol

Master curve entry

L1

READY
MEASURE PROTOCOL -OTHERS-

L2

PROTOCOL
MASTER CURVE CREATE CHANGE

L2a L3

L4a

L3a R2b

L41 L4b

L5

L48 L30

L49 L38

L1

READY
MEASURE PROTOCOL -OTHERS-

L50

SELECT TYPE OF MEASUREMENT ?
PROTOCOLS DIRECT ENTRY RATEMETER

L51

ENTER PROTOCOL NO. 7
PRINT LIST

Fig. 7-1: Flow chart showing protocol setup and measurement

Enter Master Curve

PROTOCOL NUMBER
PRINT LIST

PROTOCOL # O8 "LIA"
RAW DATA CUTOFF -OTHERS-

08: PROTOCOL NAME

08: PROTOCOL TYPE
RAW DATA CUTOFF -OTHERS-

08: PROTOCOL TYPE ?
QUANT TUP/FTI -OTHERS-

Measurements

Start of measurement

08: CHARGE

08: HIGH CAL. CONC.

LAST DISPLAY

08: INPUT ALL CORRECT ? YES
YES NO

08: OPERATOR ID.

08: FULL STANDARDIZATION YES
YES NO LAST DISPLAY

08: INPUT ALL CORRECT ? YES
YES NO

LB 9507 Chapter 7: Quantitative Measurements (LIA...)

7-3

7.2 Setting Up Quantitative Protocols

Start by entering the parameters for the measur ement procedure
and the evaluation strategy in a measurement protocol in the
<PROTOCOL> menu, and then save them. Later, you can run a
measurement simply by calling up one of these stored protocols in
the <MEASURE> menu and pressing the <START> button.

You can save up to 40 different measurement protocols which
include all information such as injector control, definition of
replicates, measuring time etc., as well as information on data
evaluation. Each new protocol is given a number which is
dependent upon the time the protocol was set up but not on the
protocol type.

The differences between the three measurement strategies are
evident in the protocol structure.

For measurem ents with a com plete standar d s eries, you will need
the entire protocol that is described on the following pages.

For measurements with a master curve the protocol finishes at
display L30 (see Fig. 7-1). The master curve is then entered in the
submenu <MASTER CURVE> and assigned to the respective
protocol.

Chapter 7: Quantitative Measurements (LIA...) LB 9507

7-4

L1

READY
MEASURE PROTOCOL -OTHERS-

Select the <PROTOCOL> menu by pressing the respective
button. Then follows:

L2

PROTOCOL
MASTER CURVE CREATE CHANGE

The <MASTER CURVE> option can be used only when a
“quantitative protocol” without a standard series has been created
and stored. If you press the button below <CHANGE> you can
open an existing protocol by entering its protocol number, and edit
it. To create a new protocol, press the button below <CREATE>.
The following display is shown:

L3

08: PROTOCOL NAME LIA-T1

The next free number is displayed for the new protocol (in this
example “08:”). Enter a name for this protocol using the
alphanumeric keyboard. You should use a name which specifies
the protocol type and other important features, so that it is easy to
identify later. For example “LIA-T1”.
Confirm your entry with <enter>.

L4a

08: PROTOCOL TYPE
RAW DATA CUTOFF -OTHERS-

Select <OTHERS>.

L4b

08: PROTOCOL TYPE
QUANT. TUP/FTI -OTHERS-

Select <QUANT>. Now you have to enter the parameters which
are the same for all protocol types:

LB 9507 Chapter 7: Quantitative Measurements (LIA...)

7-5

L5

08: OPERATOR ID.

Enter the user name and press <enter>.
If no injectors are activated (see chapter 3.8.2) the Display L15
follows.

L6

08: ENTER INJECTOR VOLUME 1 [µl]

LAST DISPLAY

Changing or confirmation of the displayed injector volume.
Displayed are the standard settings for injector 1 entered in the
<INSTRUMENT PARAMETERS> menu. These settings can be
changed for this specific protocol.
Setting "0" means the respective injector is disabled.
Range: 25 µL - 300 µL in 5 µL steps.
If the user enters the injector volume not within the 5 µL step
range, the system rounds up or down. Values out of the range are
adjusted to the limits (25 µL or 300 µL).
Example: W hen entering 43 µL the system rounds up to 45 µL,
when entering 102 µL the system rounds down to 100 µL.
Press <enter> to use the response displayed in the upper-right
corner of the LCD.

L7

08: ENTER INJECTOR VOLUME 2 [µl]

LAST DISPLAY

Changing or confirmation of the displayed injector volume.
Displayed are the standard settings for injector 2 entered in the
<INSTRUMENT PARAMETERS> menu. These settings can be
changed for this specific protocol.
Setting "0" means the respective injector is disabled.
<LAST DISPLAY> takes you back to the previous display.
Setting "0" takes you to display L10.
Setting >0 takes you to display L8.
Press <enter> to use the response displayed in the upper-right
corner of the LCD.

Chapter 7: Quantitative Measurements (LIA...) LB 9507

7-6

L8

08: SEQUENCE OF INJECTIONS 1->2
1->2 2->1 LAST DISPLAY

If you have defined 2 injectors, you have to define the s equence
of the injections here: injector 1 first and then injector 2 or vice
versa. Make your selection by pressing the button below the
option you want. Press <enter> to use the response displayed in
the upper-right corner of the LCD. The following display appears:

L9

08: DELAY TIME INJ 1/INJ 2 [s] 1.2
(0.7 - 300.0) LAST DISPLAY

Enter the desired delay in seconds between both injections and
confirm with <enter>.

L10

08: MEASURE BACKGROUND (BKG) NO
YES NO LAST DISPLAY

Select here whether the background rate is to be measured prior
to the injections.
If you select <NO>, display L14 appears or - if no injector was
selected - L15.
<YES> takes you to the next display:

L11

08: MEASURING TIME BKG [s] 0.5
(0.1 - 200.0) LAST DISPLAY

Enter between 0.1 and 200.0 seconds for the background
measurement and pres s <enter>. Reasonable times start at half
a second, allowing statistical variations to be compensated for.
Otherwise, the background values may be too high or too low
which either distorts the result of the m easurem ent or leads to an
exceeding of the threshold.

Remember that the background effect is measured in order to
obtain an alarm threshold for background rates that are too high.