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Compendium of

Background Information
COBI-CD
cobas e 411
Roche Diagnostics
2 COBI-CD Version 1.0
cobas e 411
Revision history
Order numbers
Edition notice cobas e 411 Compendium of Background Information
Intended use This CD is provided as an information source for background information regarding
the cobas e 411 analyzer The information on this CD is available in PDF-format and
requires Adobe Acrobat Reader to be installed.
Copyrights 2001-2006, Roche Diagnostics GmbH. All rights reserved.
Trademarks COBAS, COBAS C, COBAS E, ELECSYS, and LIFE NEEDS ANSWERS are
trademarks of Roche.
All other trademarks are the propery of their respective owners.
Instrument approvals The cobas e 411 analyzer meets the requirements stated in Directive 98/79/EC of the
European Parliament and the Council of the European Union (EU) on in vitro
diagnostic medical devices. Furthermore, the cobas e 411 analyzer is manufactured
and tested according to International Standard IEC 61010-1, 2nd edition, Safety
requirements for electrical equipment for measurement, control and laboratory use,
Part 1: General requirements. This International Standard is equivalent to the
national standards Underwriters Laboratories (UL) 61010-1 2nd edition for the USA,
and CAN/CSA C22.2 No. 61010-1:2004 for Canada. Compliance is demonstrated by
the following marks:
Notice to the purchaser The purchase of this product allows the purchaser to use it solely for detection by ECL
Technology for human in vitro diagnostic uses. No general patent or other license of
any kind other than this specific right of use from purchase is granted hereby. This
product may not be used by purchaser to conduct life science research and/or
Manual
Version
Template
Version
Revision date Changes
1.0 3.0
Language Order number
English 0490 5148 018
French 0490 5148 080
German 0490 5148 001
Italian 0490 5148 050
Portuguese 0490 5148 046
Spanish 0490 5148 036
Complies with the IVD (in vitro diagnostic) directive 98/79/EC.
Issued by Underwriters Laboratories, Inc. (UL) for Canada and the USA.
C US
Roche Diagnostics
COBI-CD Version 1.0 3
cobas e 411
development, patient self-testing, drug discovery and/or development or in any
veterinary, food, water or environmental testing or use.
US Pat. 5,147,806; US Pat. 5,779,976; US Pat. 6,325,973; US Pat. 5,466,416; US Pat.
5,624,637; US Pat. 5,720,922; US Pat. 5,061,445; US Pat. 5,068,088; US Pat. 5,247,243;
US Pat. 5,296,191 and corresponding patents in other countries.
Contact addresses
Manufacturer
Authorized Representative
Hitachi High-Technologies Corporation
24-14. Nishi-shimbashi. 1-chome. Minato-ku
Tokyo. 105-8717 JAPAN
Roche Diagnostics GmbH
Sandhofer Strasse 116
D-68305 Mannheim
Germany
Roche Diagnostics
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Conventions used
Visual cues are used to help you quickly locate and interpret information in this
manual. This section explains formatting conventions used in this manual.
Symbols The following symbols are used:
Abbreviations The following abbreviations are used:
Symbol Used for
a Procedural step
o List item
e Cross-reference
h Call up of screen
Note
Caution
Warning
Laser Radiation
Biohazard
Disk system specific
Rack system specific
Abbreviation Definition
A
ANSI American National Standards Institute
C
CBT Computer Based Training
CCITT Comit consultatif international tlphonique et tlgraphique
(Consultative Committee on International Telegraph and Telephone)
CE Conformit Europenne
CLAS 2 Clinical Laboratory Automation System 2
CLIA Clinical Laboratory Improvement Amendments
COBI-CD Compendium of Background Information
CSA Canadian Standards Association
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D
dBA decibel weighted against the A-frequency response curve. This curve
approximates the audible range of the human ear.
DIL diluent
E
EC European Community
ECL electrochemiluminescence
EMC electromagnetic compatibility
EN european standard
F
FIFO first in first out
H
HCFA Health Care Financing Administration
I
IEC International Electrical Commission
IS internal standard (ISE module)
IVD in vitro diagnostic directive
K
KVA kilovolt-Ampere. Unit for expressing rating of AC electrical
machinery.
L
LDL lower detection limit see analytical sensitivity
LIS laboratory information system
LLD liquid level detection
M
MSDS material safety data sheet
N
NCCLS National Committee for Clinical Laboratory Standards
P
PC/CC ProCell/CleanCell
Q
QC quality control
R
REF reference solution for ISE module
S
SD standard deviation
S/R sample/reagent
SVGA Super Video Graphics Adapter
T
TPA tripropylamine
U
UL Underwriters Laboratories Inc.
Abbreviation Definition
Roche Diagnostics
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V
VDE Verband Deutscher Elektrotechniker (association of German
electrical engineers)
Abbreviation Definition
Roche Diagnostics
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Table of contents
Revision history 2
Contact addresses 3
Conventions used 4
Table of contents 7
Mechanical theory Part A
1 Mechanical theory
Introduction A-5
Preparative operations A-6
Test protocols A-7
Assay sequence A-8
Workflow and throughput A-11
Operation flow in analysis A-13
Detailed assay sequence A-14
Dilution steps A-21
Pretreatment steps A-22
Analyzer status conditions A-23
Measurement technology Part B
2 ECL technology
ECL measuring principles B-5
Advantages of ECL technology B-10
Test principles Part C
3 Test principles
Test principles C-5
4 Reagent concept
Introduction C-15
Data transfer media C-15
Data transfer rules C-16
Reagents for cobas e 411 analyzer tests C-16
Product labeling C-18
Data links C-19
Calibration C-21
Master calibration C-22
Lot calibration C-23
Reagent pack calibration C-23
Difference between lot and reagent calibration C-24
Calibration procedures C-25
Calibration stability C-26
Calibration validation C-26
Calibration assessment C-27
Calibration of quantitative assays C-30
Calibration of qualitative assays C-33
Result calculation for qualitative assays C-33
Quality control Part D
5 Quality control concept
Control target value (first) assignment D-5
Index Part E
Index E-3
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1 Mechanical theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-3
Mechanical theory
A
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Table of contents
Mechanical theory
This chapter provides an overview of the mechanical theory of the cobas e 411
analyzer. The assay sequence and operational flow are described, as well as dilution
steps.
Introduction .................................................................................................................. 31
Preparative operations .................................................................................................. 32
Test protocols ................................................................................................................. 33
Assay sequence ............................................................................................................... 34
Run operation .......................................................................................................... 34
First incubation at 37 C ................................................................................... 34
Pipetting of additional reagent ........................................................................ 34
Second incubation at 37 C ............................................................................... 35
Pipetting of additional reagent (pretreatment assays) .................................... 35
Third incubation at 37 C (pretreatment assays) ............................................. 35
Aspirating the reaction mixture ....................................................................... 35
Cleaning the measuring cell ............................................................................. 35
Finalization ........................................................................................................ 35
Workflow and throughput ............................................................................................ 37
Effects of test combinations on throughput ......................................................... 37
9-minute tests only ............................................................................................ 37
18-minute tests only .......................................................................................... 37
Combination of 9- and 18-minute tests ........................................................... 37
27-minute tests only .......................................................................................... 38
Combination of 18- and 27-minute tests ......................................................... 38
Typical test durations .............................................................................................. 38
Operation flow in analysis ............................................................................................ 39
Detailed assay sequence ................................................................................................ 40
Preoperational steps ............................................................................................... 40
Dispensation of reagent 1, reagent 2, and sample (disk system) .......................... 41
Dispensation of reagent 1, reagent 2, and sample (rack system) .......................... 43
First incubation ....................................................................................................... 44
Microbead preparation ........................................................................................... 45
In this chapter
Chapter
1
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Table of contents
Microbead aspiration and dispensation ................................................................ 45
Second incubation ................................................................................................... 45
Preparations for the measurement process ........................................................... 46
Measurement process .............................................................................................. 46
Signal detection and conversion ............................................................................. 47
Automatic analyzer cycles ....................................................................................... 47
Dilution steps ................................................................................................................ 47
Assay with one-step dilution ............................................................................. 47
Assay with two-step dilution ............................................................................. 48
Pretreatment steps ......................................................................................................... 48
Pretreatment assay ............................................................................................. 48
Analyzer status conditions ............................................................................................ 49
A. Stop (analyzer stop) ............................................................................................ 49
A. Stop/L. Stop (analyzer stop/line stop) ............................................................... 49
A. Stop/R. Stop (analyzer stop/rack stop) .............................................................. 49
BC card scan ............................................................................................................ 49
E. Stop (emergency stop) ........................................................................................ 49
Finalization .............................................................................................................. 49
Finalization maint. .................................................................................................. 50
Initialization ............................................................................................................ 50
L. & A. reset all (line & analyzer) ............................................................................ 50
L. Stop (line stop) .................................................................................................... 50
Liquid flow cleaning ................................................................................................ 50
M. Cell preparation ................................................................................................. 50
Operation ................................................................................................................. 50
P. Stop (partial stop) ................................................................................................ 50
R. Stop (rack stop) ................................................................................................... 51
Rack clear ................................................................................................................. 51
Reagent scan ............................................................................................................ 51
S/R pipetter prime ................................................................................................... 51
S/R probe LLD volt. ................................................................................................. 51
S. Stop (sampling stop) ........................................................................................... 51
S. Stop-S. Scan ......................................................................................................... 51
Sample scan ............................................................................................................. 51
Sipper LLD volt. ...................................................................................................... 52
Sipper pipet. prime .................................................................................................. 52
Standby .................................................................................................................... 52
Stop .......................................................................................................................... 52
System reset .............................................................................................................. 52
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Introduction
Introduction
The cobas e 411 analyzer automates the immunoassay reactions utilizing
electrochemiluminescence (ECL). The individual test steps and how the system
performs the necessary procedures are described here.
e For information on ECL immunoassay reaction methods, see Chapter 2 ECL technology.
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Preparative operations
Preparative operations
Once the analyzer is switched on, the initialization process starts. During
initialization, the mechanisms are reset to their home positions.
e Figure A-1 shows the run preparation process for the cobas e 411 hardware.
Figure A-1 Run preparation process
Start
First order?
Mechanical units reset
Were reagents
exchanged?
Scan of the reagent
barcode
Counting
AssayTips and
AssayCups
Have 90 or more
minutes passed since
the last mixing?
Microbeads mixing
Is the inventory
sufficient?
Scheduling
First pipetting
Pipetting continues
Clearing the
incubator and
the AssayTip
and AssayCup
trays
Volume
check for
ProCell and
CleanCell
Alarm inventory short
(item)
45-xx-01
Preparation cycle
First sipping
Resume cycle
Sipping continues
No
Yes
Yes
Short
Enough
No
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Test protocols
Test protocols
There are 28 test protocols that can be used on the analyzer. These protocols are
predefined by Roche Diagnostics for each test and cannot be changed by the operator.
No. Step 0 Inc 0 Step 1 Inc 1 Step 2 Inc 2 Det.
0 B R1 R2 S i 1 D
1 B R1 S i 1 R2 i 2 D
2 R1 R2 S i 1 B i 2 D
3 R1 S i 1 B R2 i 2 D
4 R0 S B R1 R2 DL i 1 D
5 R0 S B R1 DL i 1 R2 i 2 D
6 R0 S R1 R2 DL i 1 B i 2 D
7 R0 S R1 DL i 1 B R2 i 2 D
8 R0 S -> DL1 R0 B R1 R2 DL i 1 D
9 R0 S -> DL1 R0 B R1 DL i 1 R2 i 2 D
10 R0 S -> DL1 R0 R1 R2 DL i 1 B i 2 D
11 R0 S -> DL1 R0 R1 DL i 1 B R2 i 2 D
12 PS S i 0 B R1 R2 i 1 D
13 PS S i 0 B R1 i 1 R2 i 2 D
14 PS S i 0 R1 R2 i 1 B i 2 D
15 PS S i 0 R1 i 1 B R2 i 2 D
16 RM S i 0 B R1 R2 DL i 1 D
17 RM S i 0 B R1 DL i 1 R2 i 2 D
18 RM S i 0 R1 R2 DL i 1 B i 2 D
19 RM S i 0 R1 DL i 1 B R2 i 2 D
20 RM S -> DL1 RM i 0 B R1 R2 DL i 1 D
21 RM S -> DL1 RM i 0 B R1 DL i 1 R2 i 2 D
22 RM S -> DL1 RM i 0 R1 R2 DL i 1 B i 2 D
23 RM S -> DL1 RM i 0 R1 DL i 1 B R2 i 2 D
24 R1 R1 i 1 D'
25 R1 R2 i 1 D'
26 R2 R2 i 1 D'
27 PS1 PS2 S i 0 R1 R2 i 1 B i 2 D'
28 PS1 PS2 S i 0 R1 i 1 B R2 i 2 D'
29 i 1 D'
... (Reserve) i 1 D'
63 i 1 D'
Table A-1 Test protocols
R1 = Reagent 1
R2 = Reagent 2
B = Beads (microbeads in the assay reagent pack)
RO = Universal diluent (special reagent pack)
PS = Pretreatment solution (for assays such as B12, Folat, and Anti-HBc)
RM = Pretreatment for IgM
S = Sample/calibrator/control
DL = Diluted sample
D = Detection with magnet drive
D' = Detection without magnet drive
I = Incubation
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Assay sequence
Assay sequence
An immunological ECL test is made up of various pipetting steps, at least one
incubation period and a measurement step. Generally at least three test components
(sample, reagent and microbeads) are pipetted into an AssayCup. After the
appropriate incubation period, the reaction mixture is aspirated into the measuring
cell where the measurement process takes place. Each of the required pipetting cycles
is performed within a defined period (42 seconds).
The number of pipetting steps, as well as the make up of the reaction mixture are
dependent on the test method (1 or 2 step test). For some methods, predilution with
diluent and/or pretreatment with a special reagent is necessary. Thus the number of
pipetting steps is increased.
The following steps apply in principle to all methods. The sequence of the individual
processes differ from test to test.
Run operation
After the appropriate test selections for patient samples are made in the software,
operation is started according to the predetermined test protocol for each assay
selected. Initially, at least one reagent (R1 or R2) and the sample or microbeads (M)
are aspirated one after another by the S/R probe. After each aspiration, the outside of
the S/R probe AssayTip is cleaned at the rinse station. The sample and reagents are
dispensed into a new AssayCup and the AssayTip is ejected into the solid waste tray.
For some tests that require sample dilution or pretreatment, diluent or pretreatment
reagent is pipetted together with sample into an AssayCup. An aliquot of the diluted/
pretreated sample is then dispensed with reagent into a second AssayCup. Therefore,
certain tests with predilution/pretreatment may require two or more AssayCups.
e For more information on dilution, see Dilution steps on page A-21.
First incubation at 37 C
The incubation times are 4.5 or 9 minutes long, depending on the test. Some tests
require only two incubation periods, whereas tests that include pretreatment can
require three incubation periods. During the incubation step(s) the immune complex
products are formed.
Pipetting of additional reagent
Some assays (usually those with more than one incubation step) require additional
reagent pipetting. As in the initial reagent pipetting step, a new AssayTip is picked up
before reagent aspiration. The S/R probe AssayTip is washed at the rinse station after
each liquid aspiration. The liquid is then dispensed into the corresponding AssayCup
where the sample and other liquids were dispensed in the first pipetting step. The
probe rises while dispensing the reaction mixture back into the AssayCup, thereby
mixing the solution and accelerating the reaction in the AssayCup. The AssayTip is
discarded into the solid waste tray when pipetting is complete.
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Assay sequence
Second incubation at 37 C
If necessary, a second incubation step (4.5 or 9 minutes) occurs.
If using a pretreatment assay, the second incubation is similar to that described above
for First Incubation at 37 C.
Pipetting of additional reagent (pretreatment assays)
For pretreatment assays, reagent pipetting is similar to that described above for
Pipetting of additional reagent occurs.
Third incubation at 37 C (pretreatment assays)
If necessary, a third incubation step (9 minutes) occurs for pretreatment assays.
Aspirating the reaction mixture
In this process, the sipper probe first aspirates ProCell (tripropylamine solution,
TPA) to prepare the measuring cell. Then, the sipper probe aspirates the reaction
mixture from the AssayCup and transfers it to the measuring cell. The sipper probe is
washed at the rinse station and ProCell is aspirated again to rinse away the unbound
constituents of reagent and sample. Next, the ECL reaction in the measuring cell
occurs.
Cleaning the measuring cell
Once the measurement is complete, the measuring cell is cleaned with CleanCell and
prepared for a new measurement process.
It takes 42 seconds (one pipetting cycle) from the aspiration of the reaction mixture
by the sipper probe until the measuring cell is filled with ProCell and ready for the
next sample.
Finalization
Thirty minutes after documentation of the last result, the sipper pipetter flushes
system water through the sipper probe, and then fills the measuring cell with ProCell
before the analyzer returns to Standby mode.
After this procedure, every 30 minutes the waste pump of the S/R rinse station runs
for 2 seconds (waste consumption approximately 12 mL). This procedure stops if the
operation switch is switched off.
e Figure A-2 shows the finalization process for the cobas e 411 hardware.
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Assay sequence
Figure A-2 Finalization process
Last sipping
Ten cycles waiting for the
new order
Gripper moves to its home
position
Cleaning the sipper
flow with system water
Pipetter prime
Pipetter end wash
Finalization
Sipper prime
Filling the sipper
nozzle with water
Standby
Filling the measuring
cell with ProCell
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Workflow and throughput
Workflow and throughput
The workflow on the cobas e 411 analyzer system is entirely sample-orientated.
Owing to the availability of a new disposable AssayTip for each test, there is no risk of
contamination. Therefore it is possible to perform assays in any sequence, thus
allowing samples to be completed one after the other.
When all assays on the system are 18-minute assays, the optimal throughput of 88
results per hour can be reached, producing a result every 42 seconds. In combination
with 9- or 27-minute assays, or in combination with two-step dilution assays, the
instrument will slow down, depending on the percentage and sequence of tests with
other incubation times.
Effects of test combinations on throughput
The various available tests have different durations. The throughput of the cobas e
411 analyzer depends upon the way in which tests of a given duration are combined,
as explained for each of the following combinations. There may be short periods of
throughput slow-down on the disk system due to the loading of a new sample disk.
Such gaps do not occur when the rack system is used, because the Roche Diagnostics/
Hitachi 5-position racks load continuously.
9-minute tests only
9-minute tests have two incubation periods, each of 4.5 minutes duration. If only 9-
minute tests are performed, the optimal throughput will always be reached regardless
of the test mixture.
All 9-minute tests follow the same time protocol. Therefore, there will be no timing
conflicts. In one 42-second cycle, the cobas e 411 will simultaneously perform S1
(first reagent pipetting), S2 (second reagent pipetting), and D (detection).
18-minute tests only
18-minute tests have two incubation periods, each of 9 minutes duration. If only 18-
minute tests are performed, the optimal throughput will always be reached regardless
of the test mixture.
All 18-minute tests follow the same time protocol. Therefore, there will be no timing
conflicts. In one 42-second cycle, the cobas e 411 will simultaneously perform S1
(first reagent pipetting), S2 (second reagent pipetting), and D (detection).
Combination of 9- and 18-minute tests
When tests of these two durations are combined, the throughput of the cobas e 411
depends on the percentage and distribution of the 9-minute tests. As a limiting factor,
it is not possible to plan the detection of two tests during one 42 second cycle. When
scheduling the first pipetting of a 9-minute test, the system has to be sure to have an
open cycle for detection 9 minutes later. Depending on the percentage and
distribution of the 9-minute assays, throughput may or may not be affected. If the
number of requested 9-minute tests is very small, larger throughput gaps will exist.
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Workflow and throughput
27-minute tests only
27-minute tests have three incubation periods, each of 9 minutes duration. If only 27-
minute assays are performed, the throughput of the cobas e 411 is reduced to 44
results per hour. Every 13 cycles, the cobas e 411 comes into a timing problem. It is
not possible to perform a S0 (pretreatment pipetting) together with a S1 (first reagent
pipetting) within one 42 second cycle. When this happens, the instrument will stand
for 13 cycles (9 minutes) until it can pipette again without conflict.
Combination of 18- and 27-minute tests
When 18- and 27-minute assays are combined, the number of gaps created depends
on the assay mix and on the exact test sequence. The gaps can vary from 1 to 13 idle
cycles (42 seconds to 9 minutes). Limiting factors are that only one detection can take
place during one 42-second cycle, and that S0 (the pretreatment step) cannot be
combined with S1 (the first reagent pipetting).
Typical test durations
e Table A-2 contains details of the duration of some typical tests. This is not a complete list
of tests, but is provided as an example.
Test 9 minutes 18 minutes 27 minutes
Thyroid TSH, T3, FT3, T4, FT4,
T-uptake, TG, Anti-TG,
Anti-TPO
Fertility hCG LH, FSH, Prolactin, Prog,
Testo, E2, HCG+beta,
Cortisol, DHEAS, SHBG
Cardiac CK-MB, Troponin T,
Myoglobin
CK-MB, Troponin T,
Myoglobin, Digoxin,
Digitoxin, proBNP
Oncology PSA, fPSA, CEA, AFP,
CA 125m CA 15-3 II,
CA 19-9, CA 72-4,
Cyfra 21-1, NSE, S100
Infectious disease HBsAg, anti-HBs,
anti-HBc IgM
(a)
anti-HAV IgM *
Anti-HAV, Anti, HBe,
HBeAg, HIV Antigen,
HIV combi
anti-HBc
Anemia Ferritin Vit B12, Folate
Diabetes c-Peptide, e-Peptide, Insulin
Bone B-CrossLaps, Total P1NP,
N-MID Osteocalcin, PTH
(b)
Other IgE
Table A-2 Durations of some typical tests
(a) 18-minute test with two-step predilution
(b) Under development
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Operation flow in analysis
Operation flow in analysis
e Figure A-3 shows a flow chart of the operational process.
(a)
Short Turn Around Time
Figure A-3 Operational process
Rerun
Assigned
Pre-start inspection
Switch on
(Initialization and Standby)
Check alarm button
Routine operation
Calibration and control
Routine or STAT
(a)
sampling
------------------------------------------------------
Results
Sampling Stop
(Finalization, Stop, and Standby)
Switch off
Maintenance
Pre-routine operation
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Detailed assay sequence
Detailed assay sequence
The mechanical process of the instrument is described below with a sandwich test,
TSH (thyroid-stimulating hormone), used as an example. This example assumes that
the reagent pack was already registered by the analyzer and does not need calibration.
All results are calculated on the basis of an existing lot calibration.
Preoperational steps
When Start is pressed from Standby mode, the following preoperational steps occur:
1. The analyzer resets all mechanisms to their respective home positions and accesses
the data disk. Next, the S/R pipetter primes the S/R probe.
2. The gripper checks for an AssayTip in position number 1 of the AssayTip trays. If
this position is empty, the gripper remembers where it last left off and checks that
position. If this position is empty, the gripper considers the whole tray empty and
the Inventory screen is updated accordingly.
3. During the AssayTip check, the S/R probe is checked for the presence of an
AssayTip. The probe moves to the AssayTip eject station and performs the
movements to eject an AssayTip. If an AssayTip is present, it is ejected.
4. After the AssayTip check is complete, the AssayCups are checked in the same
manner. During the cup check, the analyzer finishes priming the probes.
5. Next, the gripper checks the last three of the five positions on the pipetting
station. If an AssayCup is present, the analyzer goes through the following cup
disposal sequence:
a) The gripper places an AssayTip in position 1 of the pipetting station.
b) The S/R probe picks up the AssayTip, descends into the AssayCup, and
aspirates any liquid.
c) The AssayCup is discarded, while the S/R probe moves to the rinse station and
dispenses any aspirated liquid.
d) The AssayTip is washed and then discarded.
6. The gripper moves to the incubator, where it checks all 32 incubator positions. If
an AssayCup is present, the gripper moves the AssayCup to position 5 on the
pipetting station and uses the same procedure listed in step 5 to discard the
AssayCup.
7. The S/R probe AssayTip is discarded after all the incubator positions are checked.
If the analyzer is in S. Stop, the gripper remembers where it last left off and checks for an AssayTip
in that position.
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Detailed assay sequence
Dispensation of reagent 1, reagent 2, and sample (disk system)
5. The S/R probe moves from its Standby position to the R1 aspiration position.
While activating liquid level detection, the probe descends until it is 2 mm below
the reagent surface and aspirates 50 L of R1.
While the probe is aspirating R1, the gripper puts another AssayTip in position 1
of the pipetting station.
6. If the S/R probe does not detect liquid as it descends, no reagent aspiration can
occur, and an alarm is generated.
7. After R1 aspiration, the S/R probe rises and moves to the rinse station. To prevent
the aspirated R1 from coming into contact with the water in the rinse station, the
probe aspirates 10 L of air. The rinse station externally washes the AssayTip.
8. During step 7, the reagent rotor rotates until the TSH reagent pack is in the R2
position.
9. The S/R probe moves from the rinse station to the R2 aspiration position while
aspirating another 10 L of air. This air layer prevents R1 from mixing with R2.
While activating liquid level detection, the probe descends until it is 2 mm below
the reagent surface and aspirates 50 L of R2. While the probe is aspirating R2, the
gripper moves an AssayCup to position 5 of the pipetting station.
e See Figure A-4 for the location of the R2 aspiration position.
A TSH sample is present on position 1 of the sample disk.
1. After preoperational functions are complete, the gripper takes an AssayTip from
the tip tray and transports it to position 1 of the pipetting station. The gripper
returns to its Standby position.
2. The sample disk rotates until position 1 is in the sampling position.
3. The S/R probe moves to position 1 of the pipetting station, descends to obtain the
AssayTip, rises, and returns to its Standby position.
4. During this time, the reagent rotor rotates until the TSH reagent pack is at the cap
open/close mechanism. The mechanism moves forward and opens the caps on the
reagent pack. The disk rotates again to move the TSH reagent to the R1 position.
A R1 aspiration position B R2 aspiration position
Figure A-4 R1 and R2 aspiration positions
A
B
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Detailed assay sequence
10. Upon completion of R2 aspiration, the S/R probe rises and moves to the rinse
station. To prevent the aspirated R2 from coming into contact with the water in
the rinse station, the probe aspirates another 10 L of air. The rinse station
externally washes the AssayTip.
11. After R2 aspiration, the reagent rotor rotates until the TSH reagent pack is at the
cap open/close mechanism. The mechanism moves out and closes the caps.
12. The S/R probe moves from the rinse station to the sampling position while
aspirating another 10 L of air. While activating liquid level detection, the probe
descends until it is 2 mm below the sample surface and aspirates 50 L of sample.
During sample aspiration, clot detection is activated.
13. The S/R probe moves from the sampling position to position 5 of the pipetting
station. The probe descends until the tip reaches 2 mm below the calculated level
of the reaction mixture surface and dispenses the sample, R2, and R1. The probe's
downward displacement is determined by calculating the volume of the reaction
mixture for the sample and using downward-displacement tables in the software.
The probe does not rise during dispensation.
e See Figure A-5 for the location of the sampling position for disk systems.
14. After dispensation, the S/R probe moves to the tip eject position and ejects the
AssayTip.
A Sampling position
Figure A-5 Sampling position (disk system)
A
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Detailed assay sequence
Dispensation of reagent 1, reagent 2, and sample (rack system)
2. The pusher arm pushes the racks in the A-Line forward to the B-Line. The arm
returns to its home position. The first rack loads on the B-Line.
e For additional information on the A-Line and B-Line, see the Sample/reagent area
components section in the Analyzer components chapter of the cobas e 411 analyzer
Operators Manual.
3. As the rack incrementally moves on the B-Line, the rack barcode reader scans all
five rack positions and rack ID. When scanning is complete, position 1 of the rack
is in the sampling position.
4. The S/R probe moves to position 1 of the pipetting station, descends to obtain the
AssayTip, rises, and returns to its Standby position.
5. During this time, the reagent rotor rotates until the TSH reagent pack is at the
cap-open/close mechanism. The mechanism moves forward and opens the caps
on the reagent pack. The disk rotates again to move the TSH reagent to the R1
position.
e See Figure A-4 on page A-15 for details of the R1 and R2 aspiration positions.
6. The S/R probe moves from its Standby position to the R1 aspiration position.
While activating liquid level detection, the probe descends until it is 2 mm below
the surface of the reagent and aspirates 50 L of R1.
e See Figure A-4 for the location of the R1 aspiration position.
While aspirating R1, the gripper puts another AssayTip in position 1 of the
pipetting station.
7. If the S/R probe does not detect liquid during descent, no reagent aspiration can
occur, an alarm is generated.
8. After R1 aspiration, the S/R probe rises and moves to the rinse station. To prevent
the aspirated R1 from contacting the water in the rinse station, the probe aspirates
10 L of air. The rinse station externally washes the AssayTip.
9. During step 8, the reagent rotor rotates until the TSH reagent pack is in the R2
position.
10. The S/R probe moves from the rinse station to the R2 position while aspirating
another 10 L of air. This air layer prevents R1 from mixing with R2. While
activating liquid level detection, the probe descends until it is 2 mm below the
surface of the reagent and aspirates 50 L of R2. While aspirating R2 the gripper
moves an AssayCup to position 5 of the pipetting station.
e See Figure A-4 for the location of the R2 aspiration position.
11. Upon completion of R2 aspiration, the S/R probe rises and moves to the rinse
station. To prevent the aspirated R2 from coming into contact with the water in
the rinse station, the probe aspirates another 10 L of air. The rinse station
externally washes the AssayTip.
12. After R2 aspiration, the reagent rotor rotates until the TSH reagent pack is at the
cap-open/close mechanism. The mechanism moves out and closes the caps.
13. The S/R probe moves from the rinse station to the sampling position while
aspirating another 10 L of air. While activating liquid level detection, the probe
A TSH sample is present on position 1 of the rack.
1. After preoperational functions are complete, the gripper takes an AssayTip from
the tip tray and transports it to position 1 of the pipetting station. The gripper
returns to its Standby position.
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Detailed assay sequence
descends until it is 2 mm below the surface of the sample and aspirates 50 L of
sample. During sample aspiration, clot detection is activated.
14. The S/R probe moves from the sampling position to position 5 of the pipetting
station. The probe descends until the tip reaches 2 mm below where the calculated
level of the reaction mixture surface should be and dispenses the sample, R2, and
R1. The probe's downward displacement is determined by calculating the volume
of the reaction mixture for the sample and using downward-displacement tables
in the software. The probe does not rise during dispensation.
e See Figure A-6 for the location of the sampling position for rack systems.
15. After dispensation, the S/R probe moves to the tip eject position and ejects the
AssayTip.
First incubation
1. The gripper picks and transports the cup containing the reaction mixture from
the pipetting station to the incubator.
2. The cup is incubated at 37 C for 9 minutes.
3. During incubation, the analyzer continues to perform operations for other test(s)
or sample(s), if necessary.
A Sampling position
Figure A-6 Sampling position (rack system)
A
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Detailed assay sequence
Microbead preparation
Before the first incubation is completed, the TSH microbeads are mixed to facilitate
their aspiration and dispensation.
1. The reagent rotor rotates until the TSH reagent pack is at the reagent cap-open/
close mechanism. The mechanism moves out and opens the cap. The disk moves
the reagent pack to the mixing position.
2. The mixer moves over the reagent rotor and descends into the microbeads to 1.4
mm above the bottom of the bottle.
3. The mixer stirs the microbeads to obtain a homogeneous suspension.
4. During the mixing, the gripper obtains a fresh AssayTip and transports it to
position 2 of the pipetting station.
5. When mixing is complete, the mixer rises and returns to the rinse station where it
descends and rotates in the rinse station for washing.
6. At the same time, the reagent rotor rotates the TSH reagent pack to the microbead
pipetting position.
Microbead aspiration and dispensation
1. The gripper grasps the incubating cup and transports it to position 5 of the
pipetting station.
2. The S/R probe moves to the pipetting station and obtains the fresh AssayTip and
moves to the microbead pipetting position.
3. While activating the liquid level detection, the S/R probe descends below the
reagent surface and aspirates 40 L of microbeads.
4. After reagent aspiration, the S/R probe rises, moves to position 5 of the pipetting
station and descends to dispense the microbeads.
5. After dispense, the S/R probe descends further and aspirates the entire volume of
reaction mixture. The probe rises while dispensing the reaction mixture back into
the cup, thereby mixing the solution and accelerating the reaction in the cup. This
mixing takes place only once.
6. The S/R probe moves to the tip eject position and discards the AssayTip.
Second incubation
1. The gripper picks the cup containing the mixed reaction mixture and returns it to
the incubator.
2. The cup is incubated at 37 C for 9 minutes.
3. During incubation, the analyzer continues to perform operations for other test(s)
or sample(s), if necessary.
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Detailed assay sequence
Preparations for the measurement process
Before the second incubation is completed, the sipper probe aspirates ProCell into the
measuring cell to facilitate measurement.
1. The sipper probe moves from its home position to a ProCell bottle and descends
to 2 mm below the solution level and aspirates ProCell into the measuring cell. As
the probe descends, liquid level detection is activated. The sipper probe can
descend as low as 1.3 mm above the bottom of the ProCell bottle.
2. The sipper probe rises.
Measurement process
1. The gripper picks and transports the cup that has completed its second incubation
from the incubator to the aspiration station.
2. The sipper probe moves to the aspiration station and descends into the cup.
3. When the sipper probe detects the reaction mixture in the cup, it aspirates 150 L.
4. After aspiration, the sipper probe rises, aspirates 10 L of air, and moves to the
sipper rinse station to descend for rinsing.
5. The gripper grasps the cup from the aspiration station, transports it to the cup
disposal opening, and discards the cup.
6. The sipper probe is rinsed.
7. The sipper probe rises and moves to the ProCell position, descends into the bottle
and aspirates ProCell in a set aspiration/dispensation sequence. The immune
complexes are magnetically captured on the working electrode, but unbound
reagent and sample are washed away by ProCell.
e For additional information on the measuring cell, see Chapter 2 ECL technology.
8. After the bound-free separation, a voltage is applied between the working
electrode and the counter electrode. The ECL reaction is initiated and measured
by the photomultiplier.
e For additional information on binding and bound-free separation, see Chapter 3 Test
principles.
9. The sipper probe rises and moves to the CleanCell position and aspirates 20 L of
air. The probe then descends into the CleanCell bottle and aspirates reagent. This
procedure is repeated eight times. The alternatating flow of air and cleaning
solution washes the measuring cell. During this washing process, a voltage is
applied between the electrodes, which aids in the cleaning process.
10. The sipper probe moves to the sipper rinse station, aspirates 20 L of air, and
descends into the rinse station for washing.
11. Finally, the sipper probe rises and moves to the ProCell bottle. The probe
descends into the bottle and aspirates 500 L of ProCell. Next, the probe aspirates
90 L of ProCell and moves to the rinse station. At the rinse station, the probe
dispenses 35 L to flush the probe and prepare it for the next sample. During the
aspirations of the ProCell, a sequence of voltages is applied three times to prepare
the electrodes for the next measurement.
One cycle of the measurement process consumes approximately 2 mL each of ProCell
and CleanCell.
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Dilution steps
Signal detection and conversion
The measuring cell is kept at a constant 28 C throughout the measurement process.
The photomultiplier tube detects and converts the ECL signal into an electric signal
from which the cobas e 411 analyzer calculates assay results.
Automatic analyzer cycles
There are certain analyzer functions that occur automatically while the analyzer is
switched on.
o While the analyzer is in operation, the solid waste tray periodically shakes for
1.5 seconds.
o While the analyzer is in Standby, the reagent rotor turns 90 every 30 minutes.
o While the analyzer is in Standby, the rinse stations for the S/R probe and sipper
probe are switched on for 2 seconds every 30 minutes.
o Microbeads undergo a long mix when starting from Standby and then every 90
minutes until pipetting starts.
o Microbeads undergo a short mix and then a short mix every 60 minutes for each
reagent pack.
Dilution steps
The following is a description of how an assay with a dilution is performed, including
the number of AssayTips and AssayCups used in the process.
Assay with one-step dilution
(1:2, 1:5, 1:10) AssayTip 1 ~ diluent (wash)* + sample
AssayTip 1 Diluent (wash)* + sample AssayCup 1
AssayTip 2 R1 (wash)* + R2 (wash)* AssayCup 2 (1st incubation)
AssayTip 3 M (wash)* AssayCup 2 (2nd incubation)
Detection
Table A-3 Dilution steps for an assay with one-step dilution (1:2, 1:5, 1:10)
* (wash) = the outside of the AssayTip is washed.
R1 = Reagent 1
R2 = Reagent 2
M = Microbeads
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Pretreatment steps
Assay with two-step dilution
(1:50, 1:100)
Pretreatment steps
In certain test protocols, pretreatment reagent is added before R1, R2, or M, as
summarized in the following table.
Pretreatment assay
AssayTip 1 Diluent (wash)* + sample AssayCup 1
AssayTip 2 Diluent (wash)*
+ diluted sample from AssayCup 1
AssayCup 2
AssayTip 3 R1 (wash)* + R2 (wash)*
+ diluted sample from AssayCup 2
AssayCup 3
(1st incubation)
AssayTip 4 M (wash)* AssayCup 3
(2nd incubation)
Detection
Table A-4 Dilution steps for an assay with two-step dilution (1:50, 1:100)
* (wash) = the outside of the AssayTip is washed.
R1 = Reagent 1
R2 = Reagent 2
M = Microbeads
AssayTip 1 PT1 (wash)* + PT2 (wash)*
+ sample
AssayCup 1
(1st incubation)
AssayTip 2 R1 + pretreated sample in AssayCup 1 AssayCup 1
(2nd incubation)
AssayTip 3 M (wash)* + R2
+ reaction mixture in AssayCup 1
AssayCup 1
(3rd incubation)
Detection
Table A-5 Pretreatment steps for an assay
* (wash) = the outside of the AssayTip is washed.
PT1 = Pretreatment 1
PT2 = Pretreatment 2
R1 = Reagent 1
R2 = Reagent 2
M = Microbeads
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Analyzer status conditions
Analyzer status conditions
The cobas e 411 analyzer has a number of status conditions. The status conditions
usually seen during routine operation or maintenance procedures are listed below.
Several other status conditions, most them seen during various adjustment or
maintenance procedures performed by a Roche Diagnostics service representative, are
not included below.
e Refer to the Alarm screen for further information about instrument alarms.
A. Stop (analyzer stop)
A. Stop/L. Stop (analyzer stop/line stop)
A. Stop/R. Stop (analyzer stop/rack stop)
BC card scan
This status is seen when a barcode card scan is initiated from the Control Definition
or Calibration Data screens.
E. Stop (emergency stop)
An emergency stop condition exists. An alarm was issued. Take the appropriate
measures to resolve the problem.
Finalization
This is the status of the analyzer when it is between the status conditions S. Stop and
Standby.
The analyzer is no longer able to continue operation. An alarm was issued. Take the
appropriate measures to resolve the problem.
The analyzer is already in A. Stop status when the lines stop operation.
The analyzer is already in A. Stop status when the A-Line stops supplying racks to the
B-Line.
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Analyzer status conditions
Finalization maint.
This status occurs when Finalization Maintenance is initiated from the Maintenance
screen.
Initialization
This status is seen when the cobas e 411 analyzer is switched on, or when Start is
pressed from Standby.
L. & A. reset all (line & analyzer)
L. Stop (line stop)
Liquid flow cleaning
Liquid flow cleaning occurs when this function is initiated from the Maintenance
screen.
M. Cell preparation
Measuring cell (M. Cell) preparation occurs when this function is initiated from the
Maintenance screen.
Operation
This is the status during which the cobas e 411 analyzer performs its routine
operations.
P. Stop (partial stop)
A partial stop condition exists. An alarm was issued. Take the appropriate measures
to resolve the problem.
L. and A. reset all status occurs when the corresponding function is initiated from the
Maintenance screen. This function resets the analyzer and the lines.
All lines stop operation. An alarm was issued. Take the appropriate measures to
resolve the problem.
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Analyzer status conditions
R. Stop (rack stop)
Rack clear
Reagent scan
This status is seen when a reagent scan is initiated from the Inventory screen.
S/R pipetter prime
This status occurs when the S/R (sample/reagent) pipetter prime is initiated from the
Maintenance screen.
S/R probe LLD volt.
This status is seen when the analyzer is monitoring the liquid level detection voltage
of the S/R (sample/reagent) probe. The check is initiated from the Voltage Monitor
screen (Utility) folder.
S. Stop (sampling stop)
S. Stop-S. Scan
Sample scan
This status occurs when there are no more racks to process on the A-Line or B-Line.
Rack clear status occurs when the corresponding function is initiated from the
Maintenance screen. This function clears any remaining racks on the A-, B- or C-
Lines.
This status occurs when S. Stop is pressed or when sampling is complete.
The analyzer is in S. Stop and a sample scan is requested from the Status screen, or S is
pressed while the analyzer is in S. Stop.
This status occurs when a sample scan is initiated from the Status screen.
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Analyzer status conditions
Sipper LLD volt.
The analyzer is monitoring the liquid level detection voltage of the sipper probe. The
check is initiated from the Voltage Monitor screen (Utility) folder.
Sipper pipet. prime
This status occurs when the sipper pipetter prime is initiated from the Maintenance
screen.
Standby
The analyzer is not performing any operations.
Stop
This status occurs when Stop is pressed or when a Stop alarm condition exists. If an
alarm exists, take the appropriate measures to resolve the problem.
System reset
A system reset is initiated from the Maintenance screen.
2 ECL technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-3
Measurement technology
B
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Table of contents
ECL technology
This chapter provides an overview of the electrochemiluminescent technology in the
cobas e 411 analyzer system. The use of the ruthenium complex and the measuring
cell in which the reaction occurs are described.
ECL measuring principles .............................................................................................. 5
Use of the ruthenium complex ................................................................................ 5
The ECL reaction at the electrode surface ............................................................... 6
ECL signal generation ............................................................................................... 8
ECL measuring cell .................................................................................................... 9
Advantages of ECL technology ..................................................................................... 10
In this chapter
Chapter
2
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Table of contents
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COBI-CD Version 1.0 B-5
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ECL measuring principles
ECL measuring principles
Electrochemiluminescent (ECL) processes are known to occur with numerous
molecules, including compounds of ruthenium, osmium, rhenium, and other
elements.
ECL is a process in which highly reactive species are generated from stable precursors
at the surface of an electrode. These highly reactive species react with one another,
producing light.
The development of ECL/Origen immunoassays is based on the use of a
ruthenium(II)-tris(bipyridyl) [Ru(bpy)
3
]
2+
complex and tripropylamine (TPA). The
final chemiluminescent product is formed during the detection step.
e For further information on the ruthenium complex, refer to Figure B-1.
The chemiluminescent reactions that lead to the emission of light from the
ruthenium complex are triggered electrically, rather than chemically. This is achieved
by applying a voltage to the immunological complexes (including the ruthenium
complex) that are attached to streptavidin-coated microbeads. The advantage of
electrically initiating the chemiluminescent reaction is that the entire reaction can be
precisely controlled.
Use of the ruthenium complex
ECL technology uses a ruthenium chelate as the complex for the development of
light. Salts of ruthenium-tris(bipyridyl) are stable, water-soluble compounds. The
bipyridyl ligands can be readily modified with reactive groups to form activated
chemiluminescent compounds.
For the development of ECL immunoassays, a N-hydroxysuccinimide (NHS) ester of
a modified Ru(bpy)
3
complex is used because it can be easily coupled with amino
groups of proteins, haptens, and nucleic acids. This allows the detection technology
to be applied to a wide variety of analytes.
Figure B-1 The ruthenium complex
Ru
N
N
N O
O
O
N
2
+
N
N
N
O
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ECL measuring principles
The ECL reaction at the electrode surface
Two electrochemically active substances, the ruthenium complex and tripropylamine
(TPA), are involved in the reactions that lead to the emission of light. Both substances
remain stable as long as a voltage is not applied.
The ECL reaction of ruthenium-tris(bipyridyl)
2+
and TPA occurs at the surface of a
platinum electrode. The applied voltage creates an electrical field, which causes all the
materials in this field to react. TPA is oxidized at the electrode, releases an electron
and forms an intermediate TPA radical-cation, which further reacts by releasing a
proton (H
+
) to form a TPA radical (TPAo).
e For further information on the detection of a ruthenium-labeled immune complex, refer
to Figure B-2.
In turn, the ruthenium complex also releases an electron at the surface of the
electrode thus oxidizing to form the Ru(bpy)
3
3+
cation. This ruthenium cation is the
second reaction component for the following chemiluminescent reaction with the
TPA radical.
e For further information on the ECL reaction at the electrode surface, refer to Figure B-3.
Figure B-2 Detection of a ruthenium-labeled immune complex
TPA
TPA
TPA
+
-H
+
e
-
e
-
Electrode
Diffusion
Photon
Magnetic
microbead
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ECL measuring principles
TPAo and Ru(bpy)
3
3+
react with one another, whereby Ru(bpy)
3
3+
is reduced to
Ru(bpy)
3
2+
and at the same time forms an excited state through energy transfer. This
excited state is unstable and decays, with emission of a photon at 620 nm to its
original state. The reaction cycle then starts again. The tripropylamine radical reduces
to by-products that do not affect the chemiluminescence process. TPA is used up and
therefore must be present in excess. The reaction is controlled by diffusion of the TPA
and the amount of ruthenium complex present. As TPA in the electrical field is
depleted, the signal strength (light) is slowly reduced once the maximum is reached.
Although TPA is depleted during measurement, the ruthenium ground-state
complex is continually regenerated. This means that the ruthenium complex can
perform many light-generating cycles during the measurement process. This has an
inherent amplification effect that contributes to the sensitivity of the technology.
Many photons can be created from one antigen-antibody complex.
Figure B-3 The ECL reaction at the electrode surface
TPA
+
TPA
-H
+
Ru(bpy)
3
3+
e
-
e
-
e
-
Ru(bpy)
3
2+
Ru(bpy)
3
2+
TPA
Photon
(620 nm)
Electrode
surface
excited
state ground
state
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ECL measuring principles
ECL signal generation
The following figure illustrates a typical ECL signal generation. Viewed from an
electrical perspective, the reaction can be explained as follows: When a voltage is
applied to the electrode of the measuring cell, a brief peak of light emission occurs,
which can be detected as the resulting ECL signal. A defined area under the curve is
measured around the intensity maximum.
The dotted line indicates the voltage at the electrode used to generate the ECL signal.
The solid line is the actual light output measured by the photomultiplier detector.
Figure B-4 ECL signal generation
0.00
0
50,000
100,000
150,000
200,000
250,000
300,000
350,000
0.40 0.60 0.80 1.00 1.20 0.20
0
300
600
900
1200
1500
ECL intensity (counts)
Time [s]
applied voltage [mV]
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ECL measuring principles
ECL measuring cell
The core of the detection unit is the ECL measuring cell, which is designed as a flow-
through cell. The following figure shows the main components of the measuring cell:
The temperature is maintained at 28C . Three operating steps are performed in the
measuring cell:
o Bound/free separation
Using a magnet, the streptavidin microbeads that are coated with antigen-
antibody complexes are uniformly deposited on the working electrode. A system
buffer (ProCell) is used to wash the particles on the working electrode and to flush
out the excess reagent and sample materials from the measuring cell.
o ECL reaction
To initiate the ECL reaction, the magnet is removed and a voltage is applied to the
electrode. The microbeads that are coated with antigen-antibody complexes are
deposited onto the electrode. The light emission is measured with a
A Screw B Counter electrode C Optical window
D Distance washer E Top cell F Cell gap
G Gasket H O-ring I Diaphram
J Reference electrode K Outlet fitting L Working electrode
M Movable magnet N Inlet fitting O Cell body
Figure B-5 Measuring cell of the detection unit
O
N M L K
J
I
H
G
F
E D C B A
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Advantages of ECL technology
photomultiplier. The system then uses the corresponding signals for the
calculation of results.
o Release of microbeads and cell cleaning
Once the measurement is completed, the paramagnetic microbeads are washed
away from the electrode surface with a special cleaning solution (CleanCell). The
surface of the measuring cell is regenerated by varying the potential on the
electrode. The cell is then ready for another measurement.
Advantages of ECL technology
ECL (electrochemiluminescence) is an innovative technology that offers distinct
advantages over other detection techniques:
o The extremely stable nonisotopic label means that you can use convenient liquid
reagents.
o The combination of enhanced sensitivity and short incubation times leads to
high-quality assays and rapid results.
o The large measuring range, encompassing five orders of magnitude, minimizes
the need for dilutions and repeats, reducing handling time and reagent
consumption.
o The applicability of the technique to detect all analytes provides a solid platform
for menu expansion.
A Magnetic microbeads with bound antigen-
antibody complex
B Photomultiplier
C Counter electrode D Unbound antibody (ruthenium-labeled)
E Flow channel F Magnet
G Working electrode
Figure B-6 ECL measuring cell
F
D
B
A
G
C
E
3 Test principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-3
4 Reagent concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-13
Test principles
C
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Table of contents
Test principles
This chapter provides an overview of the immunology test principles used by the
cobas e 411 analyzer.
Test principles ............................................................................................................... 13
Competitive principle ............................................................................................. 14
Sandwich principle .................................................................................................. 16
Bridging principle ................................................................................................... 18
In this chapter
Chapter
3
Roche Diagnostics
COBI-CD Version 1.0 C-4
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Table of contents
Roche Diagnostics
COBI-CD Version 1.0 C-5
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Test principles
Test principles
Three test principles are available on the cobas e 411 analyzer:
o Competitive principle for extremely small analytes
o Sandwich principle (one or two steps) for larger analytes
o Bridging principle to detect antibodies in the sample
The following diagram illustrates the three available test principles:
e For detailed descriptions of these principles, see:
Competitive principle on page C-6
Sandwich principle on page C-8
Bridging principle on page C-10
Figure C-1 ECL assay principles
Sandwich principle for high
molecular weight analysis
Bridging principle to
determine IgG and IgM
Competitive principle for low
molecular weight haptens
Surface of para-
magnetic microbead
Streptavidin-biotin
binding
Analyte
Antibody
ECL label
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Test principles
Competitive principle
This principle is applied to analytes of low molecular weight, such as free
triiodothyronine (FT3).
e Refer to Figure C-2 on page C-7 for an illustration of the competitive principle.
o In the first step, sample and a specific anti-T3 antibody labeled with a ruthenium
complex are combined in an assay cup.
o After the first incubation, biotinylated T3 and streptavidin-coated paramagnetic
microbeads are added. The still-free binding sites of the labeled antibody become
occupied, with formation of an antibody-hapten complex. The entire complex is
bound to the microbeads through the interaction of biotin and streptavidin.
o After the second incubation, the reaction mixture containing the immune
complexes is transported into the measuring cell. The immune complexes are
magnetically captured on the working electrode, but unbound reagent and sample
are washed away by ProCell.
o In the ECL reaction, the conjugate is a ruthenium-based derivative and the
chemiluminescent reaction is electrically stimulated to produce light. The amount
of light produced is indirectly proportional to the amount of antigen in the
patient sample.
The concentration of the antigen is evaluated and calculated by means of a calibration
curve that was established using standards of known antigen concentration.
Roche Diagnostics
COBI-CD Version 1.0 C-7
cobas e 411 3 Test principles
Test principles
Figure C-2 Competitive principle
TPA

ECL
TPA

COMPETITIVE PRINCIPLE
FIRST REACTION
Magnetic force and
electrical potential
Signal (light)
Concentration
SECOND REACTION
LIGHT REACTION
Antigen
Biotinylated
antigen
Ruthenium-labeled
antibody
Streptavidin-coated
microbead
Tripropylamine
Roche Diagnostics
C-8 COBI-CD Version 1.0
3 Test principles cobas e 411
Test principles
Sandwich principle
The sandwich principle is applied to higher molecular weight analytes, such as
thyroid-stimulating hormone (TSH).
e Refer to Figure C-3 on page C-9 for an illustration of the sandwich principle.
o In the first step, the patient sample is combined in an AssayCup with a reagent
containing biotinylated TSH antibody and a ruthenium-labeled TSH-specific
antibody in an assay cup. During a 9-minute incubation step, antibodies capture
the TSH present in the sample.
o In the second step, streptavidin-coated paramagnetic microbeads are added.
During a second 9-minute incubation, the biotinylated antibody attaches to the
streptavidin-coated surface of the microbeads.
o After the second incubation, the reaction mixture containing the immune
complexes is transported into the measuring cell; the immune complexes are
magnetically entrapped on the working electrode, and the unbound reagent and
sample are washed away by ProCell.
o In the ECL reaction, the conjugate is a ruthenium-based derivative and the
chemiluminescent reaction is electrically stimulated to produce light. The amount
of light produced is directly proportional to the amount of TSH in the sample.
The concentration of the antigen or analyte is evaluated and calculated by means of a
calibration curve using standards of known antigen concentration.
Roche Diagnostics
COBI-CD Version 1.0 C-9
cobas e 411 3 Test principles
Test principles
Figure C-3 Sandwich principle
TPA

ECL


TPA
SANDWICH PRINCIPLE
FIRST REACTION
Magnetic force and
electrical potential
Signal (light)
Concentration
SECOND REACTION
LIGHT REACTION
Antigen
Biotinylated
antibody
Ruthenium-labeled
antibody
Streptavidin-coated
microbead
Tripropylamine
Serum constituents
Roche Diagnostics
C-10 COBI-CD Version 1.0
3 Test principles cobas e 411
Test principles
Bridging principle
The bridging principle is similar to the sandwich principle, except that the assay is
designed to detect antibodies (for example, IgG, IgM, and IgA), not antigens. This is
accomplished by including biotinylated and ruthenium-labeled antigens in the
reagents for which the targeted antibody has affinity.
e Refer to Figure C-4 on page C-11 for an illustration of the bridging principle.
o In the first step, serum antibodies bind with the biotinylated and ruthenium-
labeled antigens to form an immune complex.
o The immune complex then reacts with streptavidin-coated microbeads through
the action of the biotinylated antigen.
o After the second incubation, the reaction mixture containing the immune
complexes is transported into the measuring cell; the immune complexes are
magnetically entrapped on the working electrode, and the unbound reagent and
sample are washed away by ProCell.
o In the ECL reaction, the conjugate is a ruthenium based derivative and the
chemiluminescent reaction is electrically stimulated to produce light. The amount
of light produced is directly proportional to the amount of analyte in the sample.
The concentration of the antibody is evaluated and calculated by means of a
calibration curve that was established using standards of known antibody
concentrations.
Roche Diagnostics
COBI-CD Version 1.0 C-11
cobas e 411 3 Test principles
Test principles
Figure C-4 Bridging principle
TPA

TPA

ECL
BRIDGING PRINCIPLE
FIRST REACTION
Magnetic force and
electrical potential
Signal (light)
Concentration
SECOND REACTION
LIGHT REACTION
Biotinylated
antigen
Serum
antibodies
Tripropylamine
Serum
constituents
Streptavidin-coated
microbeads
Ruthenium-
labeled antigen
Roche Diagnostics
C-12 COBI-CD Version 1.0
3 Test principles cobas e 411
Test principles
Roche Diagnostics
COBI-CD Version 1.0 C-13
cobas e 411 4 Reagent concept
Table of contents
Reagent concept
This chapter provides an overview of all types of reagents used on the cobas e 411
analyzer system. It describes the various reagent containers used, and also provides an
overview of the system-related reagent management, explaining processes such as
how the system registers new reagents, and how it monitors reagent consumption.
Introduction ................................................................................................................. 15
Data transfer media ....................................................................................................... 15
Data transfer rules ......................................................................................................... 16
Reagents for cobas e 411 analyzer tests ........................................................................ 16
Diluents .................................................................................................................... 16
System reagents ........................................................................................................ 17
Calibrators and controls .......................................................................................... 17
Reagent packs .......................................................................................................... 17
Product labeling ............................................................................................................ 18
Data links ...................................................................................................................... 19
Calibration .................................................................................................................... 21
Master calibration ......................................................................................................... 22
Lot calibration ............................................................................................................... 23
Reagent pack calibration ............................................................................................... 23
Difference between lot and reagent calibration .......................................................... 24
Calibration procedures ................................................................................................ 25
Calibration stability ....................................................................................................... 26
Calibration validation ................................................................................................... 26
Calibration assessment .................................................................................................. 27
Missing values .......................................................................................................... 27
Monotony of curve (quantitative assays only) ....................................................... 27
Slope (qualitative assays only) ................................................................................ 27
Calibration factor (quantitative assays only) ......................................................... 28
Minimum signal ...................................................................................................... 29
Minimum/maximum signal (qualitative assays only) .......................................... 29
Minimum difference (quantitative assays only) ................................................... 29
Minimum acceptable difference (qualitative assays only) .................................... 29
In this chapter
Chapter
4
Roche Diagnostics
COBI-CD Version 1.0 C-14
cobas e 411 4 Reagent concept
Table of contents
Deviation of duplication ......................................................................................... 29
System errors ........................................................................................................... 29
Cal. .......................................................................................................................... 30
Target (quantitative assays only) ............................................................................ 30
Cutoff (qualitative assays only) ............................................................................... 30
Borderline (qualitative assays only) ........................................................................ 30
Calibration of quantitative assays ................................................................................. 30
Rodbard function .................................................................................................... 31
Linear calibration function ..................................................................................... 32
Linear reciprocal calibration function ................................................................... 32
Calibration of qualitative assays ................................................................................... 33
Result calculation for qualitative assays ....................................................................... 33
Roche Diagnostics
COBI-CD Version 1.0 C-15
cobas e 411 4 Reagent concept
Introduction
Introduction
Elecsys reagent packs (cobas e packs) have a special 2D (two-dimensional) barcode.
This allows fully automatic registration and management of reagent information. The
advantage of this barcode is that manual entry or additional monitoring is not
necessary. The ready-to-use liquid reagents are loaded into one of the 18 positions on
the reagent rotor. Reagents are available for analysis after their barcodes are scanned.
The handling of calibrators and Roche Diagnostics controls is similar to that of
reagents. Some calibrators are supplied ready to use. Lyophilized controls and some
calibrators must be prepared and transferred into the appropriate container. For
quantitative assays, calibrator and control information is stored on 2D barcode cards.
For qualitative assays, all information necessary for calibration is encoded on the
barcoded labels.
Data transfer media
The following data sources are available for cobas e 411 analyzer applications:
1. Barcodes on reagent packs (2D matrix barcode)
o Reagent pack
o Diluent reagent pack
o Pretreatment reagent pack
o BlankCell
2. Barcodes on calibrator and controls vials (1D barcode)
o Calibrator primary vial
o Control primary vial
3. cobas Link and system database (created during installation)
o Assay
o Calibrator and barcode card
o Control and barcode card
Barcodes on calibrator and controls vials carry information such as calibrator or
control identification, level number, and lot ID.
Reagent pack barcodes and downloaded data encode a lot more information,
including application codes, calibration validation criteria, and expiry dates.
e For further information of information encoded onto the barcode labels, see Data links on
page C-19.
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Data transfer rules
Data transfer rules
Calibrator data source Calibrator data are encoded on the 2D barcode of the reagent pack.
o If the reagent pack was produced after the CalSet (calibrator set), the target values
from the reagent pack have priority and are used for generating the calibration
curve.
o If the reagent pack was produced before the CalSet, data followed by target values
from the calibrator card are used for generating the calibration curve.
Control target values from
different data sources
Control target values are provided by different data sources.
If a target value for a control for a specific PreciControl lot/reagent lot combination
has been manually, the analyzer uses this value rather than the value read from the
control barcode card or reagent barcode. When determining which control target
value to use, the analyzer applies the following priority rules:
Priority 1: Target values entered manually for a specific reagent lot
Priority 2: Target values read from the reagent barcode
Priority 3: Target vaules read from the control barcode card (or data downloaded
from cobas Link - under development)
If a new reagent lot or control lot is then placed on the analyzer, it uses the control
values encoded in the barcodes for these new lots.
Reagents for cobas e 411 analyzer tests
This section describes all reagents necessary to run the cobas e 411 analyzer, and the
reagents that are specific for each available test. The available tests are classified into
different groups:
o Thyroid
o Fertility
o Cardiac
o Oncology
o Infectious disease
o Anemia
o Diabetes
o Bone
o Other
e For more information, see User access levels in the Software description section of the
online Help.
Diluents
For most tests where dilution may be necessary, use Universal Diluent or MultiAssay
as diluent. However, some testsincluding Anti-HAV, Estradiol, Progesterone, and
NSE (neuron-specific enolase )require specific diluents.
Roche Diagnostics
COBI-CD Version 1.0 C-17
cobas e 411 4 Reagent concept
Reagents for cobas e 411 analyzer tests
e For information on required diluents and recommended dilution factors, refer to the
package inserts of the particular assay reagents.
System reagents
The cobas e 411 analyzer uses the following system reagents:
Calibrators and controls
There are specific calibrators for each test. As for quality controls, there are both
controls covering multiple tests and controls that are specific for only a single test.
e For information on required calibrators and controls, refer to the respective package
insert.
To get information about what calibrator and controls are currently needed for
calibration or QC, print a Calibration/QC Load List from the software.
Reagent packs
The principal reagent container for the cobas e 411 is the cobas e pack.
e For additional information on the packaging and barcoding of reagents, see the Product
labeling section in the System overview chapter of the cobas e 411 analyzer Operators
Manual.
Reagent Use Bottle size
ProCell o Conditioning of the electrodes
o Transport of the assay reaction mixture
o Washing of the streptavidin-coated microbeads
o Signal generation
380 mL
CleanCell o Cleaning of the tubing system and of the measuring cell after every measurement
o Conditioning of the electrodes
380 mL
SysClean o Sodium hypochlorite solution used for cleaning of the measuring cells (every two weeks).
o SysClean is not stored on the instrument.
100 mL
SysWash o Increasing rinsing efficiency between pipetting steps.
o Avoiding reagent carryover.
o Preventing bacterial growth.
o SysWash is added to the distilled water container, with a dilution 1+100.
500 mL
Table C-1 System reagents for the cobas e 411 analyzer.
Roche Diagnostics
C-18 COBI-CD Version 1.0
4 Reagent concept cobas e 411
Product labeling
A cobas e pack is a reagent pack that consists of three separate, capped reagent
containers. The cobas e 411 can open and close these caps automatically. There is an
individual reagent pack available for each test.
Product labeling
Each reagent pack is equipped with a barcode label. The barcode label contains
reagent, control, and calibration information. The reagent barcode labels are in a
unique format. The symbology uses portable data files (PDF) and is called PDF417.
Traditional linear barcodes serve as a link to relevant information stored in a
database. However a PDF417 is a two-dimensional, stacked barcode encoded to
contain an entire data record. The large amount of data that can be encoded allows all
instrument settings to be included, as well as the master calibration curve and
additional information for the assay. From this master calibration curve and from the
operator 2-point calibration, the analyzer derives the update of the master calibration
curve.
Every PDF417 symbol (barcode) contains two error detection codewords that are
used like the check digit in linear barcode symbologies to detect decode errors and
verify that all data has been read and decoded accurately. Additionally, PDF417
provides error correction in the event that portions of the symbol have been
damaged, destroyed or are unreadable.
(a)
It is a combination of this error detection and error correction that ensures a reliable
barcode. There should only be a small number of exceptional cases when barcodes are
so badly damaged that the analyzer cannot read them. If the barcode cannot be read
Figure C-5 Reagent pack
(a) 1. Itkin S, Martell J. A PDF417 Primer: A Guide to Understanding Second Generation Barcodes and
Portable Data Files. Bohemia, NY: Symbol Technologies, Inc; 1992:17-18.
Roche Diagnostics
COBI-CD Version 1.0 C-19
cobas e 411 4 Reagent concept
Data links
and the reagent lot has previously been used by the analyzer, you can manually enter
the 15-digit number found on the reagent barcode label into the software.
Data links
This following table illustrates the information that may be encoded on the barcode
labels. Background colors are used to indicate calibration links that exist between
information on separate barcodes.
The exact information encoded onto the barcode labels is not detailed here, as this
information is propriety to Roche Diagnostics. Items that are not directly linked are
mapped within the cobas e 411 software or user interface.
Roche Diagnostics
C-20 COBI-CD Version 1.0
4 Reagent concept cobas e 411
Data links
e For further information about reagent checklists, see Reagents, calibrators, and controls in
the Troubleshooting chapter of the cobas e 411 analyzer Operators Manual.
Reagent Pack
barcode
Calibrator
barcode card
Calibrator vial
barcode
Control
barcode card
Control vial
barcode
Diluent
barcode
Test number Test number Test number Test number
(space for 28
different tests (test
number, target
values and ranges
in %)
Test number
Lot number:
calibrator (space
for 5 different
calibrator control
values)
Lot number:
calibrator
Lot number:
calibrator
Lot number:
reagent pack
Lot number:
reagent pack (space
for 10 different
reagent pack lots
and calibrator
target values)
Reagent pack:
bottle number
Calibrator vial
number
Control vial
number
Test lot ID
(only for ID-assays
or assays where
calibrators are
within the reagent
pack package)
Test lot ID Test lot ID Test lot ID Test lot ID
Calibrator levels Calibrator level
number
Control level
number
Lot number:
control (space for
10 different control
lot target values)
Lot number:
control
Lot number:
control
Rodbard
parameters
Calibration
validation criteria
Calibrator
identification
Control
identification
Control number Control number
Expiry date Expiration date Expiration date Expiration date
Table C-2 Calibration barcode information
Roche Diagnostics
COBI-CD Version 1.0 C-21
cobas e 411 4 Reagent concept
Calibration
Calibration
Calibration is required to determine the concentration of an unknown substance as
accurately as possible. For this, a master calibration curve is generated at Roche
Diagnostics during production of the reagent and is encoded in the 2D barcode of the
appropriate reagent pack. This information is then transferred to the analyzer. At the
customer site, the analyzer generates an update of the master curve by measuring two
calibrators under routine laboratory conditions.
The calibration curve produced from the barcoded master calibration and the
measured calibration is specific to each reagent lot and, in some cases, to an
individual reagent pack. The condition of the analyzer and the reagents influence the
system-specific calibration. The result of a calibration is validated automatically by
the analyzer and can be further validated by the operator.
Figure C-6 Calibration procedure
Master Calibration
performed by Roche Diagnostic
Data Carrier Calibration
with two calibrators
Roche Diagnostics
C-22 COBI-CD Version 1.0
4 Reagent concept cobas e 411
Master calibration
Master calibration
The following diagram illustrates how measurement accuracy can be increased by
combining the calibration results from a lot calibration of master calibrators
produced by Roche Diagnostics (RD) with the information encoded in the 2D
barcode:
A reference standardization curve utilizing master test kit reagents and certified
reference standard material [for example, World Health Organization (WHO)
reference material] is measured at Roche Diagnostics. This curve uses 10 to 12 points
(n = 10 to 12). The reference standard curve is the basis for the production of master
calibrators.
A lot-specific master calibration curve (n = 5 or 6) is measured at Roche Diagnostics
using lot-specific test kit reagents and master calibrators. The shape of the lot-specific
master curve is characterized by a four-parameter Rodbard function. The data
characterizing this curve are stored in the lot-specific reagent barcode. Lot-specific
calibrator assigned values (CalSet assigned values) are read off the lot-specific master
calibration curve and are encoded in the barcode label of the reagent pack.
At the customer site, the calibration results from two calibrators that were measured
under routine conditions are mathematically combined with the encoded data from
the 2D barcode. From this combination, the system determines a lot calibration or
reagent pack calibration from which the concentration of measured samples is
reliably calculated.
Figure C-7 Calibration concept

RD Development
RD Production
Customer
E
f
f
o
r
t
A
c
c
u
r
a
c
y
Roche Diagnostics
COBI-CD Version 1.0 C-23
cobas e 411 4 Reagent concept
Lot calibration
Lot calibration
A lot calibration (L-Calib) is a calibration performed with a fresh reagent pack that
has not been on the analyzer longer than 24 hours and for which all calibration
validation criteria are acceptable. Reagent-specific calibrators are used to update two
of the four Rodbard curve-defining parameters. This adjusts the curve to match the
original lot-specific calibration curve.
The lot calibration is valid for all other reagent packs of the same lot, provided these
reagent packs were stored as specified in the package insert and have not been on the
analyzer for longer than seven days.
e For further information, see Calibration factor (quantitative assays only) on page C-28.
Reagent pack calibration
A reagent pack calibration (R-Calib) is performed with a reagent that has been on the
analyzer for more than 24 hours.
A reagent pack calibration is valid for one specific reagent pack only. The reagent
pack calibration is compared to the most recent stored L-Calib for validation.
Roche Diagnostics
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4 Reagent concept cobas e 411
Difference between lot and reagent calibration
Difference between lot and reagent calibration
The following diagram illustrates the process for determining whether a lot or reagent
pack calibration is required:
Figure C-8 Overview of the lot and reagent pack calibration process
Check samples and
then repeat calibration
Not all calibration criteria
acceptable
Calibration is not released
Reagent pack
Controls and samples after
operator release will use this
reagent pack calibration
Controls and samples before
operator release will use the
previous lot calibration
All calibration criteria
acceptable
Automatic system release
Reagent pack calibration
(system-released)
Valid for this reagent pack only
Controls and samples after
system release will use this
reagent pack calibration
On analyzer < 24 hours On analyzer > 24 hours
All calibration criteria
acceptable
Automatic system release
Calibration
Controls and
samples
Lot calibration
Valid for all reagent packs of the
same lot
Roche Diagnostics
COBI-CD Version 1.0 C-25
cobas e 411 4 Reagent concept
Calibration procedures
Calibration procedures
The following table contains examples of the procedures to be followed for various
scenarios when more than one reagent pack is used for one assay:
Example case Procedure
Four reagent packs (same lot):
o One reagent pack (old, previously used
reagent pack, on analyzer < 24 h)
o Three reagent packs (new, on analyzer <
24 h)
Controls are measured daily for each reagent pack. If a control is measured out of
range for all four reagent packs:
o Check the registration time, because a lot calibration may be necessary.
o Request a manual calibration for all four reagent packs: a new lot calibration
cannot be used, because one reagent pack has already been used. Each reagent
pack is assigned its own lot calibration.
The most recently performed lot calibration is used.
The calibrated reagent packs will be controlled if a control rack is placed in the
input buffer directly after the calibrator rack (control of calibration).
Samples will be pipetted with the current, old reagent pack.
Three reagent packs (same lot):
o One reagent pack (old, previously used
reagent pack, on analyzer > 24 h)
o Two reagent packs (new, on analyzer >
24 h)
Controls are measured daily for each reagent pack. If a control is measured out of
range for all of the reagent packs:
o Check the registration time, as a lot calibration may be necessary.
o It is recommended to place a new reagent pack, so that a lot calibration can be
carried out.
o Request a manual calibration for all four reagent packs (the three reagent packs
that were already placed and the one that is newly placed): a new lot calibration
cannot be used, because one reagent pack has already been used. Each reagent
pack is assigned its own reagent pack calibration and the new reagent pack will
get a lot calibration when all calibration criteria are within the specifications.
Three reagent packs (same lot):
o One reagent pack (old, previously used
reagent pack, on analyzer > 24 h)
o Two reagent packs (new, on analyzer >
24 h)
Controls are measured daily for each reagent pack. If a control is measured out of
range for one of the new reagent packs, the reagent pack is calibrated and
controlled. This reagent pack is assigned a reagent pack calibration as the reagent
pack has been on board > 24 h.
Three reagent packs (same lot):
o One reagent pack (old, previously used
reagent pack, on analyzer > 24 h)
o Two reagent packs (new, on analyzer <
24 h)
Controls are measured daily for each reagent pack. If a control is measured out of
range for one of the new reagent packs, the reagent pack is calibrated and
controlled. This reagent pack is assigned a lot calibration (if all calibration criteria
are within the limits) as the reagent pack has been on board for < 24 h.
Table C-3 Example cases and procedures
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C-26 COBI-CD Version 1.0
4 Reagent concept cobas e 411
Calibration stability
Calibration stability
The stability of calibration is determined by two factors:
o The long-term stability of the instrumentation
o The age of the reagent
For many assays, a reagent pack will be used within seven days. In this situation, it is
not necessary to renew the calibration for the new reagent pack. In this case, the lot
calibration can be used for all other new reagent packs for a period as recommended
in the Calibration Frequency section of the package insert. After that period, a new lot
calibration is recommended.
If the reagent is kept on the analyzer for more than seven days, it is recommended that
the calibration be renewed. This renewal of the calibration can be repeated as needed
until the on-analyzer open stability of the reagent is exceeded (for example, two
months).
Calibration validation
The calibration of a test can be easily identified on the Calibration > Status screen.
If the test is highlighted in red, this indicates that both of the calibrator measurements
have failed or the calibration factor is outside the range 0.9-1.2 . There is no red
highlighting if the calibration was successful.
Figure C-9 Calibration workflow
new lot: lot calibration
(2 points, obligatory)
high throughput
t
i
m
e
new reagent pack is opened
start
calibration recomended
every 28 days
renewed calibration (2 points)
recommended after 7 days
same reagent pack is used
over several weeks
low throughput
Calibration can be performed more frequently if required. This may be because of local regulations,
or before performing specific types of test.
Roche Diagnostics
COBI-CD Version 1.0 C-27
cobas e 411 4 Reagent concept
Calibration assessment
Calibration assessment
The quality criteria of a calibration are displayed on the Working Information
window in the Calibration menu.
All calibrations are automatically checked for:
o Missing values
o Monotony of curve
o Slope
o Calibration factor
o Minimum/maximum signal
o Minimum acceptable difference
o Deviation of duplication
o System error
o Calibrator measurement signals
o Target
o Cutoff
o Borderline
Missing values
Duplicate determinations of two calibrators are used to adjust the master calibration
curve stored on the reagent pack barcode. Therefore, you must have a minimum of
n-1 values for all calibrator replicates measured (n = total number of calibrator
replicates. For any current Elecsys assay, this number totals 4.). Currently, all Elecsys
reagents use only two calibrators. This box can accommodate up to five calibrators.
Check to see if any alarms occurred during calibration that may have caused the
missing values. Treat any questionable calibration according to laboratory policy.
Monotony of curve (quantitative assays only)
All measured calibrator values must fall in ascending (sandwich or bridging
principle) or descending (competition principle) order. This is termed monotony.
This box displays five dashes representing up to five calibrators. If either a 1 (Cal 1) or
a 2 (Cal 2) is displayed in this box, the result is a failed calibration.
Slope (qualitative assays only)
All measured calibrator values must fall in ascending (sandwich or bridging
principle) or descending (competition principle) order. If this is not the case, or the
slope is less than or greater than the slope encoded in the reagent barcode, the
calibration fails. The slope of the assay is listed as OK or NG (Not Good).
Roche Diagnostics
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4 Reagent concept cobas e 411
Calibration assessment
Calibration factor (quantitative assays only)
A curve position check against the most recent lot calibration produces a calibration
factor. This box displays a number that represents this factor.
Each new lot calibration (L-Calib) uses a calibration factor of 1. For all subsequent
reagent pack calibrations (R-Calib), a new calibration factor is calculated. The
calibration factor is the quotient of the slopes of the actual performed calibration and
the related stored calibration.
The calibration factor is set to 1 at each new lot calibration. The following reagent
pack calibrations are compared with the last measured lot calibration. The calibration
factor is the ratio between the calibrator signals (difference of CalSet 1 and CalSet 2)
of the lot and reagent pack calibration. The calibration factor is only used as a
calibration validation criteria and not used for sample calculation.
The following formulae show the relationship between lot calibration and reagent
pack calibration:
The calibration factor criterion is used only in validating reagent pack calibration (R-Cal).
Example:
Calibration factor for each Lot calibration
t
1
t
1
---- 1 = =
t
1
CalSet 1 signal (standardization) CalSet 2 signal (standardization)
actual CalSet 1
l
signal actual CalSet 2
l
signal
------------------------------------------------------------------------------------------------------------------------------------------------------------------------ =
t
r
CalSet 1 signal (standardization) CalSet 2 signal (standardization)
actual CalSet 1
r
signal actual CalSet 2
r
signal
------------------------------------------------------------------------------------------------------------------------------------------------------------------------ =
Calibration factor for Reagent pack calibration
t
1
t
r
----
actual CalSet 1
r
signal actual CalSet 2
r
signal
CalSet 1
l
signal CalSet 2
l
signal
--------------------------------------------------------------------------------------------------------------------------- = =
TSH
1000 22000 counts
1100 25000 counts
--------------------------------------------------- 0.88 = =
Reagent calibration
Lot calibration
r
l
This simplified formula is only valid when the same calibrator concentrations are used for the
reagent pack and lot calibration. If these calibrator concentrations are different, the calibration
signals of the standardization have to be considered.
Roche Diagnostics
COBI-CD Version 1.0 C-29
cobas e 411 4 Reagent concept
Calibration assessment
Minimum signal
The measured signal of the calibrator replicate must be above the minimum value.
Values are test dependent and are encoded in the reagent barcode. Currently, all
Elecsys reagents use only two calibrators. This table can accommodate up to five
calibrators.
Check to see if any alarms occurred during calibration that may have caused a
calibrator replicate to have an unacceptable minimum signal. Treat any questionable
calibration according to your laboratory policy.
Minimum/maximum signal (qualitative assays only)
The measured signal of the calibrator should fall between the designated minimum
and maximum signals. Minimum and maximum signals are test dependent and are
encoded in the reagent barcode. If all calibrator replicates were sampled with no
errors, this box displays four dashes, representing the calibrator replicates.
Minimum difference (quantitative assays only)
Defined as the difference in percent of the values between calibrator 1 and 2. This
difference must amount to at least 30% for the calibration to be accepted.
Minimum acceptable difference (qualitative assays only)
The difference between the negative and positive calibrator signal values must be
greater than the allowable value limit. This limit is test dependent and is encoded in
the reagent barcode. The minimum acceptable difference is listed as OK or NG (Not
Good).
Deviation of duplication
The deviation of duplicate measurements is a check of the signal values for each
replicate of a calibrator. If the difference between the duplicate measurements is too
great, the appropriate calibrator is flagged. The signal values are used to calculate the
mean value of the duplicate measurements.
System errors
A hardware error occurred during a calibrator measurement. If either 1 (Cal 1) or 2
(Cal 2) is displayed in this box, the result is a failed calibration.
Roche Diagnostics
C-30 COBI-CD Version 1.0
4 Reagent concept cobas e 411
Calibration of quantitative assays
Cal.
The actual measurement signal levels of Cal 1 and Cal 2, used to calculate the mean
value of the duplicate measurements. Two measurements are taken:
1. Signal The actual signal level of the first measurement of Cal 1 or Cal 2. The mean of the first
and second measurements is used in the calculation of the calibration curve.
2. Signal The actual signal level of the second measurement of Cal 1 or Cal 2. The mean of the
first and second measurements is used in the calculation of the calibration curve.
Target (quantitative assays only)
The target value of the calibrator is encoded in the 2D barcode of the reagent pack.
Cutoff (qualitative assays only)
Qualitative assays are calibrated by a scaling factor, or the cutoff value. The actual
cutoff value is calculated by means of the cutoff formula on the basis of at least one
reactive or nonreactive calibrator. Each sample receives a scaled result value, the
cutoff value, which allows for the classification of samples being reactive or
nonreactive.
Borderline (qualitative assays only)
For some assays it is possible that in a range around a Cutoff Index = 1, no
determination regarding reactive or nonreactive results can be made. This range is
called the borderline or borderline area.
Calibration of quantitative assays
The following is a description of the different methods utilized by the cobas e 411
analyzer for calculating results. To calculate quantitative tests, the system uses the
following three calibration functions to convert measured signals into
concentrations:
o Rodbard function
o Linear calibration function
o Linear reciprocal calibration function
The calibration function used by the system is encoded in the 2D barcode on the
appropriate reagent pack. The calculations are performed automatically by the
analyzer, including the correction for samples diluted by the analyzer.
Roche Diagnostics
COBI-CD Version 1.0 C-31
cobas e 411 4 Reagent concept
Calibration of quantitative assays
Rodbard function
The relation between the measured signal and the corresponding analyte
concentration is given the following equation:
Equation C-1
Parameters b and c define the shape of the curve and parameters a and d define the
position of the curve.
Under the controlled conditions of automation on the analyzer, the shape of the
calibration curve is very stable and, therefore, it is possible to calibrate this nonlinear
function with only two calibrators and the information of the shape parameters b and
c. The curve position parameters a and d are calculated with each calibration. Such a
calibration is called 2-point calibration.
The following inverse formula is used to determine the sample concentration based
on its signal.
Equation C-2
Sample concentration
Rodbard function parameters
Signal
Signal
Rodbard function parameters
Sample concentration
y
a d
1
x
b
---


c
+
-------------------- d + =
x
a b c d , , ,
y
x b
a y
y d
-----------

1 c /
=
y
a b c d , , ,
x
Roche Diagnostics
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4 Reagent concept cobas e 411
Calibration of quantitative assays
Linear calibration function
The relation between the measured signal and the corresponding analyte
concentration is given the following equation:
Equation C-3
Calibrations using a linear calibration curve are always performed using two
calibrators.
The following inverse formula is used to determine the sample concentration based
on its signal.
Equation C-4
Linear reciprocal calibration function
The relation between the measured signal and the corresponding analyte
concentration is given the following equation:
Equation C-5
Calibrations using a linear reciprocal calibration curve are always performed using
two calibrators.
The following inverse formula is used to determine the sample concentration based
on its signal.
Equation C-6
Signal
Concentration
Calibration curve parameters (y-intercept and slope)
Sample concentration
Calibration curve parameters
Signal
y b x a + =
y
x
a b ,
x
y a
b
----------- =
x
a b ,
y
Signal
Concentration
Calibration curve parameters (y-intercept and slope)
Sample concentration
Calibration curve parameters
Signal
1
y
--- b x a + =
y
x
a b ,
x
1 a y
b y
------------------ =
x
a b ,
y
Roche Diagnostics
COBI-CD Version 1.0 C-33
cobas e 411 4 Reagent concept
Calibration of qualitative assays
Calibration of qualitative assays
In order to assess patient samples as reactive, nonreactive, or borderline, a cutoff
value is calculated.
Two calibrators, reactive (REAC) and nonreactive (N-REAC), are used for
calibration. These calibrators produce effective signals and from which
the cutoff value is calculated as follows.
Equation C-7
Result calculation for qualitative assays
In order to calculate the result of a qualitative assay (cutoff test), the system compares
the effective signal of the measurement with the cutoff signal of the calibration
. For that purpose, a cutoff index is calculated as the ratio between
the effective signal and the cutoff signal as follows.
Equation C-8
If the effective signal of the measurement equals the cutoff signal of the
calibration , the cutoff index equals 1. For effective signals being
lower or higher than the cutoff signal, the cutoff index is smaller or larger than 1,
respectively.
In order to evaluate the reactivity of a sample, the 2D barcode contains defined limit
values. If the cutoff indices, which were calculated from the effective signals, lie
between the lower limit (LL) and the upper limit (UL), no decision can be made
regarding the reactivity or non-reactivity of the sample (borderline).
The test result is evaluated as follows, depending on the test principle (sandwich tests
show a positive slope, and competitive tests show a negative slope).
Cutoff value
Effective signal of the reactive calibrator
Effective signal of the nonreactive calibrator
Assay-specific cutoff parameters
(according to the 2D barcode)
S
Cutoff
S
POS
S
NEG
S
Cutoff
A S
NEG
B S
POS
C + + =
S
Cutoff
S
POS
S
NEG
A B C , ,
Cutoff index
Effective signal of sample measurement
Cutoff value of the calibrator
S
eff
S
Cutoff
Cutoff
I ndex
Cutoff
I ndex
S
eff
S
Cutoff
----------------- =
Cutoff
I ndex
S
eff
S
Cutoff
S
eff
S
Cutoff
Cutoff
I ndex
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4 Reagent concept cobas e 411
Result calculation for qualitative assays
The analyzer automatically calculates the cutoff based on the measurement of Cal1
and Cal2. The results of a sample is given either as reactive, nonreactive, or
borderline, as well as in the form of a cutoff index.
For sandwich assays:
o Samples with a cutoff index > 1.0 are considered to be reactive.
o Samples with a cutoff index < 1.0 are considered to be nonreactive.
For some assays a grey zone is introduced.
For competitive assays:
o Samples with a cutoff index > 1.0 are considered to be nonreactive.
o Samples with a cutoff index < 1.0 are considered to be reactive.
Result Sandwich test (positive slope) Competitive test (negative slope)
Reactive
Nonreactive
Borderline
Table C-4 Qualitative assay result evaluation
Cutoff
I ndex
UL Cutoff
I ndex
LL
Cutoff
I ndex
UL < Cutoff
I ndex
UL >
LL Cutoff
I ndex
UL < LL Cutoff
I ndex
UL <
5 Quality control concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-3
Quality control
D
Roche Diagnostics
COBI-CD Version 1.0 D-3
cobas e 411 5 Quality control concept
Table of contents
Quality control concept
This chapter provides a brief overview of the assignment of control target values for
the cobas e 411 analyzer.
Control target value (first) assignment ........................................................................ 33
Roche Diagnostics quality control principles ........................................................ 33
Control rules and specifications ............................................................................. 33
In this chapter
Chapter
5
Roche Diagnostics
COBI-CD Version 1.0 D-4
cobas e 411 5 Quality control concept
Table of contents
Roche Diagnostics
COBI-CD Version 1.0 D-5
cobas e 411 5 Quality control concept
Control target value (first) assignment
Control target value (first) assignment
Roche Diagnostics quality control principles
A current reagent lot is measured with a new manufactured Roche Diagnostics
control. The control value is written on the control barcode card independent of the
reagent lot.
This control value is valid until a specific target value for a reagent lot exists.
Control rules and specifications
Procedure: four instruments, with two runs each
A reagent will be standardized against the master lot (master reagent and master
calibrator) with all valid calibrators available.
All valid Roche Diagnostics controls are measured and checked for deviations.
Case 1 All controls are within the target range: < +/- 1 SD
The target values of the existing control barcode cards are used for this reagent lot,
which means, the control values are not changed when using this reagent lot.
Case 2 The controls are out of the target range: > +/- 1 SD for this reagent lot
The target values of the controls are on the reagent pack barcode, which means the
specific target values for reagent lots exist and the control values on the control card
are not valid. As a consequence, an extra information sheet is put inside the reagent
kit indicating the re-assigned values and the new values are stated on the reagent pack
barcode.
If the difference between the medians of the target values for cobas e 411 analyzer controls is less
than 1 SD, the mean of these medians is used as the target value.
Advantage The target value of the control is identical.
Disadvantage The recovered value can have another level within the given range.
For example, 105% for reagent lot 1 and 94% for reagent lot 2.
Advantage The control value will be recovered to approximately 100%.
Disadvantage A specific control value for the reagent lot exists.
o Priority 1: Manually entered control target values
o Priority 2: Reagent pack 2D barcode
o Priority 3: Control barcode card or data downloaded from cobas Link (under development)
This means once a target value of a control is entered manually this value is valid as long as the
customer uses a new control lot. If the customer deletes this control on the QC Install screen and
scans the control barcode card once again, then the manual input is no longer valid.
Roche Diagnostics
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5 Quality control concept cobas e 411
Control target value (first) assignment
Due to the priority rules with a new reagent lot, the target value of a control will not
be taken from the control barcode card or the reagent barcode if the target value has
been entered manually for this assay.
The main point of each standardization action is to receive the same human serum
recovery independent of the reagent lot. Multi-analyte controls are spiked, stripped
and preserved and unfortunately, do not always react in the same way.
Index
E
Roche Diagnostics
COBI-CD Version 1.0 E-3
cobas e 411 Index
Index
Abbreviations, 4
Analyzer cycles
automatic, A-21
Approvals, instrument, 2
Aspiration
reaction mixture, A-9
Assay principles
bridging principle, C-10
competitive principle, C-6
sandwich principle, C-8
Assay sequence, A-8, A-14
Barcode
calibration information, C-19
Borderline range, C-30
Bridging principle, C-10
Calibration
barcode information, C-19
curve, C-21
data links, C-19
factor, C-28
introduction, C-21
master, C-22
procedures, C-25
qualitative assays, C-33
quality criteria, C-27
quantitative assays, C-30
reagent pack, C-23, C-24
signal level, C-30
stability, C-26
validation, C-26
Calibration assessment, C-27
Calibration quality criteria
borderline, C-30
calibration factor, C-28
calibration signal level, C-30
cutoff, C-30
deviation of duplicate, C-29
minimum acceptable difference in calibrator signal,
C-29
minimum difference, C-29
minimum signal, C-29
minimum/maximum signal, C-29
missing values, C-27
monotony, C-27
slope, C-27
system errors, C-29
target, C-30
Cleaning
measuring cell, A-9
Competitive principle, C-6
Contact addresses, 3
Control target value assignment
Roche controls, D-5
Copyrights, 2
Cutoff value, C-30
Data links
calibration, C-19
Deviation of duplicate calibration measurement, C-29
ECL (electrochemiluninescence)
advantages of, B-10
assay principles, C-5
measuring cell, B-9
measuring principles, B-5
reaction, B-6
signal generation, B-8
Finalization, A-9
Flow
operation, A-13
Function
linear calibration, C-32
linear reciprocal calibration, C-32
Rodbard, C-31
Immunology calibration
calibration validation, C-26
lot calibration, C-23, C-24
master calibration, C-22
procedures, C-25
qualitative assays, C-33
quality criteria, C-27
quantitative assays, C-30
reagent pack calibration, C-23, C-24
stability, C-26
Incubation
first, A-8, A-18
second, A-9, A-19
third, A-9
Initialization process, A-6
Instrument
approvals, 2
A
B
C
D
E
F
I
Roche Diagnostics
E-4 COBI-CD Version 1.0
Index cobas e 411
Intended use, 2
Labeling
product, C-18
License, 2
Linear calibration function, C-32
Linear reciprocal calibration function, C-32
Lot calibration, C-23
Master calibration, C-22
Master calibration curve, C-21
Measurement
preparations, A-20
reaction mixture, A-9
Measurement process, A-20
Measuring cell
cleaning, A-9
description, B-9
Measuring principles
ECL (electrochemiluninescence), B-5
Microbead
aspiration, A-19
dispensation, A-19
preparation, A-19
Minimum acceptable difference in calibrator signal,
C-29
Minimum calibrator signal, C-29
Minimum difference in calibrator signal, C-29
Minimum/maximum signal, C-29
Missing calibration values, C-27
Monotony of calibration curve, C-27
Operation flow, A-13
Operators Manual
conventions used, 4
version, 2
Patents, 2
Preoperational steps, A-14
Product labeling, C-18
Protocols
test, A-7
Qualitative assays
calibration, C-33
result calculation, C-33
Reaction mixture
aspiration, A-9
measurement, A-9
Reagent concept
introduction, C-15
Reagent pack calibration, C-23, C-24
Reagent pipetting
additional, A-8
pretreatment assays, A-9
Result calculation
qualitative assays, C-33
Rodbard function, C-31
Ruthenium complex, B-5
Sandwich principle, C-8
Scaling factor, C-30
Sequence
assay, A-8
Signal detection and conversion, A-21
Slope of calibration curve, C-27
Software
version, 2
Symbols, 4
System errors during calibrator measurement, C-29
Target value of calibrator, C-30
Test principles
bridging principle, C-10
competitive principle, C-6
overview, C-5
sandwich principle, C-8
Test protocols, A-7
Throughput
effects of test combinations, A-11
workflow, A-11
Trademarks, 2
TSH microbeads
preparation, A-19
Workflow, A-11
L
M
O
P
Q
R
S
T
W