cobas e 411 COBI-CD

Compendium of Background InformationCOBI-CD

cobas e 411

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

2 COBI-CD · Version 1.0

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

COBI-CD · Version 1.0 3

cobas e 411

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 Comité consultatif international téléphonique et télégraphique

(Consultative Committee on International Telegraph and Telephone)

CE Conformité Européenne’

CLAS 2 Clinical Laboratory Automation System 2

CLIA Clinical Laboratory Improvement Amendments

COBI-CD Compendium of Background Information

CSA Canadian Standards Association

Roche Diagnostics


cobas e 411

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.


Roche Diagnostics


cobas e 411

V

VDE Verband Deutscher Elektrotechniker (association of German

electrical engineers)


Roche Diagnostics


cobas e 411

Table of contents

Revision history 2Contact addresses 3Conventions used 4Table of contents 7

Mechanical theory Part A

1 Mechanical theoryIntroduction A-5Preparative operations A-6Test protocols A-7Assay sequence A-8Workflow and throughput A-11Operation flow in analysis A-13Detailed assay sequence A-14Dilution steps A-21Pretreatment steps A-22Analyzer status conditions A-23

Measurement technology Part B

2 ECL technologyECL measuring principles B-5Advantages of ECL technology B-10

Test principles Part C

3 Test principlesTest principles C-5

4 Reagent conceptIntroduction C-15Data transfer media C-15Data transfer rules C-16Reagents for cobas e 411 analyzer tests C-16Product labeling C-18Data links C-19Calibration C-21Master calibration C-22Lot calibration C-23Reagent pack calibration C-23Difference between lot and reagent calibration C-24Calibration procedures C-25Calibration stability C-26Calibration validation C-26Calibration assessment C-27Calibration of quantitative assays C-30Calibration of qualitative assays C-33Result calculation for qualitative assays C-33

Quality control Part D

5 Quality control conceptControl target value (first) assignment D-5

Index Part E

Index E-3

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cobas e 411

Roche Diagnostics


1 Mechanical theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-3

Mechanical theory A

cobas e 411 1 Mechanical theoryTable 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

Roche Diagnostics

COBI-CD · Version 1.0 A-3

cobas e 411 1 Mechanical theoryTable 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

Roche Diagnostics


cobas e 411 1 Mechanical theoryIntroduction

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.

Roche Diagnostics


1 Mechanical theory cobas e 411Preparative 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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A-6 COBI-CD · Version 1.0

cobas e 411 1 Mechanical theoryTest 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

Roche Diagnostics


1 Mechanical theory cobas e 411Assay 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.

Roche Diagnostics


cobas e 411 1 Mechanical theoryAssay 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.

Roche Diagnostics


1 Mechanical theory cobas e 411Assay 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

Roche Diagnostics


cobas e 411 1 Mechanical theoryWorkflow 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.

Roche Diagnostics


1 Mechanical theory cobas e 411Workflow 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

Roche Diagnostics


cobas e 411 1 Mechanical theoryOperation 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

Roche Diagnostics


1 Mechanical theory cobas e 411Detailed 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.

Roche Diagnostics


cobas e 411 1 Mechanical theoryDetailed 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 µL 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 µL 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 µL 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 µL 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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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 µL 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 µL of air. While activating liquid level detection, the probe descends until it is 2 mm below the sample surface and aspirates 50 µL 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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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

Operator’s 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 µL of R1.


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 µL 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 µL 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 µL of R2. While aspirating R2 the gripper moves an AssayCup to position 5 of the pipetting station.


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 µL 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 µL 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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descends until it is 2 mm below the surface of the sample and aspirates 50 µL 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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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 µL 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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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 µL.

4. After aspiration, the sipper probe rises, aspirates 10 µL 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 µL 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 µL 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 µL of ProCell. Next, the probe aspirates 90 µL of ProCell and moves to the rinse station. At the rinse station, the probe dispenses 35 µL 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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cobas e 411 1 Mechanical theoryDilution 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 ú Diluent (wash)* + sample ú AssayCup 1

AssayTip 2 ú R1 (wash)* + R2 (wash)* ú AssayCup 2 (1st incubation)

AssayTip 3 ú M (wash)* ú 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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1 Mechanical theory cobas e 411Pretreatment 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 ú Diluent (wash)* + sample ú AssayCup 1

AssayTip 2 ú Diluent (wash)*

+ diluted sample from AssayCup 1

ú AssayCup 2

AssayTip 3 ú R1 (wash)* + R2 (wash)*

+ diluted sample from AssayCup 2

ú AssayCup 3

(1st incubation)

AssayTip 4 ú M (wash)* ú AssayCup 3

(2nd incubation)

Detection

Table A-4 Dilution steps for an assay with two-step dilution (1:50, 1:100)


R1 = Reagent 1

R2 = Reagent 2

M = Microbeads

AssayTip 1 ú PT1 (wash)* + PT2 (wash)*

+ sample

ú AssayCup 1

(1st incubation)

AssayTip 2 ú R1 + pretreated sample in AssayCup 1 ú AssayCup 1

(2nd incubation)

AssayTip 3 ú M (wash)* + R2

+ reaction mixture in AssayCup 1

ú AssayCup 1

(3rd incubation)

Detection

Table A-5 Pretreatment steps for an assay


PT1 = Pretreatment 1

PT2 = Pretreatment 2

R1 = Reagent 1

R2 = Reagent 2

M = Microbeads

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cobas e 411 1 Mechanical theoryAnalyzer 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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1 Mechanical theory cobas e 411Analyzer 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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cobas e 411 1 Mechanical theoryAnalyzer 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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1 Mechanical theory cobas e 411Analyzer 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.

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2 ECL technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-3

Measurement technology B

cobas e 411 2 ECL technologyTable 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


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COBI-CD · Version 1.0 B-3

cobas e 411 2 ECL technologyTable of contents

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cobas e 411 2 ECL technologyECL 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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2 ECL technology cobas e 411ECL 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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B-6 COBI-CD · Version 1.0


TPAo and Ru(bpy)33+ 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)33+

e-

e-

e-

Ru(bpy)32+

Ru(bpy)32+

TPA•

Photon (620 nm)

Electrode surface

excited stateground

state

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2 ECL technology cobas e 411ECL 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

0.000

50,000

100,000

150,000

200,000

250,000

300,000

350,000

0.40 0.60 0.80 1.00 1.200.20

0

300

600

900

1200

1500

ECL intensity (counts)

Time [s]

applied voltage [mV]

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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 28°C . 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

EDCBA

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2 ECL technology cobas e 411Advantages 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

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3 Test principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-3

4 Reagent concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-13

Test principles C

cobas e 411 3 Test principlesTable 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


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COBI-CD · Version 1.0 C-3

cobas e 411 3 Test principlesTable of contents

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cobas e 411 3 Test principlesTest 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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3 Test principles cobas e 411Test 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.

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C-6 COBI-CD · Version 1.0


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

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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.

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Figure C-3 Sandwich principle

TPA ECL

TPA

SANDWICH PRINCIPLE

FIRST REACTION

Magnetic force and


Signal (light)

Concentration

SECOND REACTION

LIGHT REACTION

Antigen

Biotinylated

antibody

Ruthenium-labeled

antibody

Streptavidin-coated

microbead

Tripropylamine

Serum constituents

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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.

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Figure C-4 Bridging principle

TPA

TPA

ECL

BRIDGING PRINCIPLE

FIRST REACTION

Magnetic force and


Signal (light)

Concentration

SECOND REACTION

LIGHT REACTION

Biotinylated

antigen Serum

antibodies

Tripropylamine

Serum

constituents

Streptavidin-coated

microbeadsRuthenium-

labeled antigen

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Roche Diagnostics


cobas e 411 4 Reagent conceptTable 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


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cobas e 411 4 Reagent conceptTable 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


cobas e 411 4 Reagent conceptIntroduction

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.

Roche Diagnostics


4 Reagent concept cobas e 411Data 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 fromdifferent 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 tests—including Anti-HAV, Estradiol, Progesterone, and NSE (neuron-specific enolase )—require specific diluents.

Roche Diagnostics


cobas e 411 4 Reagent conceptReagents 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 Operator’s

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


4 Reagent concept cobas e 411Product 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


cobas e 411 4 Reagent conceptData 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


4 Reagent concept cobas e 411Data links

e For further information about reagent checklists, see Reagents, calibrators, and controls in

the Troubleshooting chapter of the cobas e 411 analyzer Operator’s Manual.

Reagent Pack barcode

Calibrator barcode card

Calibrator vial barcode

Control barcode card

Control vial barcode

Diluentbarcode

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


cobas e 411 4 Reagent conceptCalibration

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


4 Reagent concept cobas e 411Master 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

Effo

rt

Acc

urac

y

Roche Diagnostics


cobas e 411 4 Reagent conceptLot 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


4 Reagent concept cobas e 411Difference 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


cobas e 411 4 Reagent conceptCalibration 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):


reagent pack, on analyzer > 24 h)

o Two reagent packs (new, on analyzer >

24 h)


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.




o Two reagent packs (new, on analyzer >

24 h)


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.




o Two reagent packs (new, on analyzer <

24 h)


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

Roche Diagnostics


4 Reagent concept cobas e 411Calibration 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

tim

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


cobas e 411 4 Reagent conceptCalibration 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


4 Reagent concept cobas e 411Calibration 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 calibrationt1t1---- 1= =

t1CalSet 1 signal (standardization) CalSet 2 signal (standardization)–

actual CalSet 1l signal actual CalSet 2l signal –------------------------------------------------------------------------------------------------------------------------------------------------------------------------=

trCalSet 1 signal (standardization) CalSet 2 signal (standardization)–

actual CalSet 1r signal actual CalSet 2r signal –------------------------------------------------------------------------------------------------------------------------------------------------------------------------=

Calibration factor for Reagent pack calibrationt1tr----

actual CalSet 1r signal actual CalSet 2r signal –CalSet 1l signal CalSet 2l 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


cobas e 411 4 Reagent conceptCalibration 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


4 Reagent concept cobas e 411Calibration 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


cobas e 411 4 Reagent conceptCalibration 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


y a d–

1 xb---⎝ ⎠⎛ ⎞ c

+-------------------- d+=

x

a b c d,,,

y

x b a y–y d–-----------⎝ ⎠⎛ ⎞⋅

1 c/=

y

a b c d,,,

x

Roche Diagnostics


4 Reagent concept cobas e 411Calibration of quantitative assays

Linear calibration function


Equation C-3

Calibrations using a linear calibration curve are always performed using two calibrators.


Equation C-4

Linear reciprocal calibration function


Equation C-5

Calibrations using a linear reciprocal calibration curve are always performed using two calibrators.


Equation C-6

Signal

Concentration

Calibration curve parameters (y-intercept and slope)


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)


Calibration curve parameters

Signal

1y--- b x⋅ a+=

y

x

a b,

x 1 a y⋅–b y⋅

------------------=

x

a b,

y

Roche Diagnostics


cobas e 411 4 Reagent conceptCalibration 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)

SCutoff

SPOS SNEG

SCutoff A SNEG⋅ B SPOS⋅ C+ +=

SCutoff

SPOS

SNEG

A B C,,

Cutoff index

Effective signal of sample measurement

Cutoff value of the calibrator

SeffSCutoff CutoffIndex

CutoffIndex

Seff

SCutoff-----------------=

CutoffIndex

Seff

SCutoff

SeffSCutoff CutoffIndex

Roche Diagnostics


4 Reagent concept cobas e 411Result 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

CutoffIndex UL≥ CutoffIndex LL≤

CutoffIndex UL< CutoffIndex UL>

LL CutoffIndex UL<≤ LL CutoffIndex UL≤<

Roche Diagnostics


5 Quality control concept . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-3

Quality control D

cobas e 411 5 Quality control conceptTable 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


Roche Diagnostics

COBI-CD · Version 1.0 D-3

cobas e 411 5 Quality control conceptTable of contents

Roche Diagnostics


cobas e 411 5 Quality control conceptControl 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


5 Quality control concept cobas e 411Control 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.

Roche Diagnostics

D-6 COBI-CD · Version 1.0

Index E

cobas e 411 Index

Index

Abbreviations, 4Analyzer cycles– automatic, A-21Approvals, instrument, 2Aspiration– reaction mixture, A-9Assay principles– bridging principle, C-10– competitive principle, C-6– sandwich principle, C-8Assay sequence, A-8, A-14

Barcode– calibration information, C-19Borderline range, C-30Bridging 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-26Calibration assessment, C-27Calibration 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-9Competitive principle, C-6Contact addresses, 3Control target value assignment– Roche controls, D-5Copyrights, 2Cutoff value, C-30

Data links– calibration, C-19Deviation 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-9Flow– operation, A-13Function– 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-26Incubation– first, A-8, A-18– second, A-9, A-19– third, A-9Initialization process, A-6Instrument– approvals, 2

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COBI-CD · Version 1.0 E-3

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Intended use, 2

Labeling– product, C-18License, 2Linear calibration function, C-32Linear reciprocal calibration function, C-32Lot calibration, C-23

Master calibration, C-22Master calibration curve, C-21Measurement– preparations, A-20– reaction mixture, A-9Measurement process, A-20Measuring cell– cleaning, A-9– description, B-9Measuring principles– ECL (electrochemiluninescence), B-5Microbead– aspiration, A-19– dispensation, A-19– preparation, A-19Minimum acceptable difference in calibrator signal, C-29Minimum calibrator signal, C-29Minimum difference in calibrator signal, C-29Minimum/maximum signal, C-29Missing calibration values, C-27Monotony of calibration curve, C-27

Operation flow, A-13Operator’s Manual– conventions used, 4– version, 2

Patents, 2Preoperational steps, A-14Product labeling, C-18Protocols– test, A-7

Qualitative assays– calibration, C-33– result calculation, C-33

Reaction mixture– aspiration, A-9– measurement, A-9

Reagent concept– introduction, C-15Reagent pack calibration, C-23, C-24Reagent pipetting– additional, A-8– pretreatment assays, A-9Result calculation– qualitative assays, C-33Rodbard function, C-31Ruthenium complex, B-5

Sandwich principle, C-8Scaling factor, C-30Sequence– assay, A-8Signal detection and conversion, A-21Slope of calibration curve, C-27Software– version, 2Symbols, 4System errors during calibrator measurement, C-29

Target value of calibrator, C-30Test principles– bridging principle, C-10– competitive principle, C-6– overview, C-5– sandwich principle, C-8Test protocols, A-7Throughput– effects of test combinations, A-11– workflow, A-11Trademarks, 2TSH microbeads– preparation, A-19

Workflow, A-11

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