Вы находитесь на странице: 1из 89

cobas e 411

cobas e 411 Compendium of Background Information COBI-CD

Compendium of Background Information

COBI-CD

cobas e 411

Revision history

Manual Version

Template Version

Revision date

Changes

1.0

1.1

3.0

3.0

April 2011

Normal range for calibration factor corrected in "Calibration Validation" section

Order numbers

Language

Order number

English

0490 5148 018 0490 5148 080 0490 5148 001 0490 5148 050 0490 5148 046 0490 5148 036

 

French

German

Italian

Portuguese

Spanish

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

© 2001-2011, 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:

C ® Roche Diagnostics
C
®
Roche Diagnostics

Complies with the IVD (in vitro diagnostic) directive 98/79/EC.

Issued by Underwriters Laboratories, Inc. (UL) for Canada and the USA.

US

cobas e 411

Notice to the purchaser

Contact addresses

Manufacturer

Authorized Representative

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 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.

Pat. 5,296,191 and corresponding patents in other countries. Hitachi High-Technologies Corporation 24-14.

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

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:

Symbol

Used for

a

Procedural step

o

List item

e

Cross-reference

h

Call up of screen

Note

Note

Caution

Caution

Warning e Cross-reference h Call up of screen Note Caution Laser Radiation Biohazard Disk system specific Rack

Laser Radiation Cross-reference h Call up of screen Note Caution Warning Biohazard Disk system specific Rack system specific

Biohazard h Call up of screen Note Caution Warning Laser Radiation Disk system specific Rack system specific

Disk system specific up of screen Note Caution Warning Laser Radiation Biohazard Rack system specific Abbreviations The following

Rack system specific Warning Laser Radiation Biohazard Disk system specific Abbreviations The following abbreviations are used:

Abbreviations

The following abbreviations are used:

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

Abbreviation

Definition

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

I

Health Care Financing Administration

IEC

International Electrical Commission

IS

internal standard (ISE module)

IVD

in vitro diagnostic directive

K

KVA

L

kilovolt-Ampere. Unit for expressing rating of AC electrical machinery.

LDL

lower detection limit see analytical sensitivity

LIS

laboratory information system

LLD

liquid level detection

M

MSDS

N

NCCLS

P

material safety data sheet

National Committee for Clinical Laboratory Standards

PC/CC

ProCell/CleanCell

Q

QC

quality control

R

REF

S

SD

S/R

SVGA

T

TPA

U

UL

reference solution for ISE module

standard deviation sample/reagent Super Video Graphics Adapter

tripropylamine

Underwriters Laboratories Inc.

Roche Diagnostics

cobas e 411

Abbreviation

Definition

V

VDE

Verband Deutscher Elektrotechniker (association of German electrical engineers)

Roche Diagnostics

cobas e 411

Table of contents

Revision history Contact addresses Conventions used Table of contents

2

3

4

7

Mechanical theory

Part

A

1 Mechanical theory

Quality control

Part

D

5

Quality control concept

Control target value (first) assignment

D-5

Index

Part

E

Introduction

A-5

Index

E-3

Preparative operations

A-6

Test protocols

A-7

Assay sequence

A-8

Workflow and throughput

A-11

Operation flow in analysis

A-13

Detailed assay sequence

A-14

Dilution steps

A-21

Pretreatment steps

A-22

Analyzer status conditions

A-23

Measurement technology

Part

B

2 ECL technology

ECL measuring principles

B-5

Advantages of ECL technology

B-10

Test principles

Part

C

3 Test principles

Test principles

C-5

4 Reagent concept

Introduction

C-15

Data transfer media

C-15

Data transfer rules

C-16

Reagents for cobas e 411 analyzer tests

C-16

Product labeling

C-18

Data links

C-19

Calibration

C-21

Master calibration

C-22

Lot calibration

C-23

Reagent pack calibration

C-23

Difference between lot and reagent calibration

C-24

Calibration procedures

C-25

Calibration stability

C-26

Calibration validation

C-26

Calibration assessment

C-27

Calibration of quantitative assays

C-30

Calibration of qualitative assays

C-33

Result calculation for qualitative assays

C-33

Roche Diagnostics

cobas e 411

Roche Diagnostics

Mechanical theory

A

1 Mechanical theory

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

A-3

cobas e 411

1 Mechanical theory

Table of contents

Mechanical theory

This chapter provides an overview of the mechanical theory of the cobas e 411 analyzer. The assay sequence and operational flow are described, as well as dilution steps.

In this chapter

Chapter

1

Introduction

5

Preparative operations

6

Test protocols

7

Assay sequence

8

Run operation

8

First incubation at 37 °C

8

Pipetting of additional reagent

8

Second incubation at 37 °C

9

Pipetting of additional reagent (pretreatment assays)

9

Third incubation at 37 °C (pretreatment assays)

9

Aspirating the reaction mixture

9

Cleaning the measuring cell

9

Finalization

9

Workflow and throughput

11

Effects of test combinations on throughput

11

9-minute tests only

11

18-minute tests only

11

Combination of 9- and 18-minute tests

11

27-minute tests only

12

Combination of 18- and 27-minute tests

12

Typical test durations

12

Operation flow in analysis

13

Detailed assay sequence

14

Preoperational steps

14

Dispensation of reagent 1, reagent 2, and sample (disk system)

15

Dispensation of reagent 1, reagent 2, and sample (rack system)

17

First incubation

18

Microbead preparation

19

Roche Diagnostics

1 Mechanical theory

cobas e 411

Table of contents

 

Microbead aspiration and dispensation Second incubation Preparations for the measurement process Measurement process Signal detection and conversion Automatic analyzer cycles Dilution steps Assay with one-step dilution Assay with two-step dilution Pretreatment steps Pretreatment assay Analyzer status conditions

19

19

20

20

21

21

21

21

22

22

22

23

A. Stop (analyzer stop)

23

A. Stop/L. Stop (analyzer stop/line stop)

23

A. Stop/R. Stop (analyzer stop/rack stop)

23

BC card scan

23

E.

Stop (emergency stop)

23

Finalization

23

Finalization

24

Initialization

24

L.

& A. reset all (line & analyzer)

24

L.

Stop (line stop)

24

Liquid flow cleaning M. Cell preparation Operation

24

24

24

P. Stop (partial stop)

24

R.

Stop (rack stop)

25

Rack clear

25

Reagent scan

25

S/R pipetter prime S/R probe LLD

25

25

S.

Stop (sampling stop)

25

S.

Stop-S. Scan

25

Sample scan Sipper LLD Sipper pipet. prime Standby Stop System reset

25

26

26

26

26

26

Roche Diagnostics

cobas e 411

1 Mechanical theory

Introduction

Introduction

The cobas e 411 analyzer automates the immunoassay reactions utilizing electrochemiluminescence (ECL). The individual test steps and how the system performs the necessary procedures are described here.

e For information on ECL immunoassay reaction methods, see Chapter 2 ECL technology.

Roche Diagnostics

1 Mechanical theory

cobas e 411

Preparative operations

Preparative operations

Once the analyzer is switched on, the initialization process starts. During initialization, the mechanisms are reset to their home positions.

e Figure A-1 shows the run preparation process for the cobas e 411 hardware.

Start First order? Mechanical units reset Were reagents exchanged? No Yes Counting AssayTips and Scan
Start
First order?
Mechanical units reset
Were reagents
exchanged?
No
Yes
Counting
AssayTips and
Scan of the reagent
barcode
AssayCups
Volume
check for
ProCell and
CleanCell
Have 90 or more
Clearing the
No
minutes passed since
incubator and
the last
mixing?
the AssayTip
and AssayCup
Yes
trays
Microbeads mixing
Is the inventory
sufficient?
Alarm inventory short
(item)
Short
45-xx-01
Enough
Preparation cycle
Scheduling
First sipping
First pipetting
Resume cycle
Pipetting continues
Sipping continues

Figure A-1

Run preparation process

Roche Diagnostics

cobas e 411

1 Mechanical theory

Test protocols

Test protocols

There are 28 test protocols that can be used on the analyzer. These protocols are predefined by Roche Diagnostics for each test and cannot be changed by the operator.

No.

Step 0

Inc 0

 

Step 1

Inc 1

Step 2

Inc 2

Det.

0

   

B R1 R2 S

i 1

   

D

1

     

B R1 S

i 1

 

R2

i 2

D

2

     

R1 R2 S

i 1

 

B

i 2

D

3

     

R1 S

i 1

B

R2

i 2

D

4

R0 S

 

B

R1 R2 DL

i 1

   

D

5

R0 S

   

B

R1 DL

i 1

 

R2

i 2

D

6

R0 S

 

R1 R2 DL

i 1

 

B

i 2

D

7

R0 S

   

R1 DL

i 1

B

R2

i 2

D

8

R0 S -> DL1 R0

 

B

R1 R2 DL

i 1

   

D

9

R0 S -> DL1 R0

   

B

R1 DL

i 1

 

R2

i 2

D

10

R0 S -> DL1 R0

 

R1 R2 DL

i 1

 

B

i 2

D

11

R0 S -> DL1 R0

   

R1 DL

i 1

B

R2

i 2

D

12

PS S

i 0

 

B

R1 R2

i 1

   

D

13

PS S

i 0

 

B R1

i 1

 

R2

i 2

D

14

PS S

i 0

 

R1 R2

i 1

 

B

i 2

D

15

PS S

i 0

 

R1

i 1

B

R2

i 2

D

16

RM S

i 0

B

R1 R2 DL

i 1

   

D

17

RM S

i 0

 

B

R1 DL

i 1

 

R2

i 2

D

18

RM S

i 0

R1 R2 DL

i 1

 

B

i 2

D

19

RM S

i 0

 

R1 DL

i 1

B

R2

i 2

D

20

RM S -> DL1 RM

i 0

B

R1 R2 DL

i 1

   

D

21

RM S -> DL1 RM

i 0

 

B

R1 DL

i 1

 

R2

i 2

D

22

RM S -> DL1 RM

i 0

R1 R2 DL

i 1

 

B

i 2

D

23

RM S -> DL1 RM

i 0

 

R1 DL

i 1

B

R2

i 2

D

24

     

R1 R1

i 1

   

D'

25

     

R1 R2

i 1

   

D'

26

     

R2 R2

i 1

   

D'

27

PS1 PS2 S

i 0

 

R1 R2

i 1

 

B

i 2

D'

28

PS1 PS2 S

i 0

 

R1

i 1

B

R2

i 2

D'

29

     

i 1

   

D'

       

(Reserve)

i 1

   

D'

63

     

i 1

   

D'

 

Table A-1

Test protocols

R1

= Reagent 1

R2

= Reagent 2

B = Beads (microbeads in the assay reagent pack)

RO = Universal diluent (special reagent pack)

PS = Pretreatment solution (for assays such as B12, Folat, and Anti-HBc)

RM = Pretreatment for IgM

S = Sample/calibrator/control

DL = Diluted sample

D = Detection with magnet drive

D' = Detection without magnet drive I = Incubation

Roche Diagnostics

1 Mechanical theory

cobas e 411

Assay sequence

Assay sequence

An immunological ECL test is made up of various pipetting steps, at least one incubation period and a measurement step. Generally at least three test components (sample, reagent and microbeads) are pipetted into an AssayCup. After the appropriate incubation period, the reaction mixture is aspirated into the measuring cell where the measurement process takes place. Each of the required pipetting cycles is performed within a defined period (42 seconds).

The number of pipetting steps, as well as the make up of the reaction mixture are dependent on the test method (1 or 2 step test). For some methods, predilution with diluent and/or pretreatment with a special reagent is necessary. Thus the number of pipetting steps is increased.

The following steps apply in principle to all methods. The sequence of the individual processes differ from test to test.

Run operation

After the appropriate test selections for patient samples are made in the software, operation is started according to the predetermined test protocol for each assay selected. Initially, at least one reagent (R1 or R2) and the sample or microbeads (M) are aspirated one after another by the S/R probe. After each aspiration, the outside of the S/R probe AssayTip is cleaned at the rinse station. The sample and reagents are dispensed into a new AssayCup and the AssayTip is ejected into the solid waste tray.

For some tests that require sample dilution or pretreatment, diluent or pretreatment reagent is pipetted together with sample into an AssayCup. An aliquot of the diluted/ pretreated sample is then dispensed with reagent into a second AssayCup. Therefore, certain tests with predilution/pretreatment may require two or more AssayCups.

e For more information on dilution, see Dilution steps on page A-21.

First incubation at 37 °C

The incubation times are 4.5 or 9 minutes long, depending on the test. Some tests require only two incubation periods, whereas tests that include pretreatment can require three incubation periods. During the incubation step(s) the immune complex products are formed.

Pipetting of additional reagent

Some assays (usually those with more than one incubation step) require additional reagent pipetting. As in the initial reagent pipetting step, a new AssayTip is picked up before reagent aspiration. The S/R probe AssayTip is washed at the rinse station after each liquid aspiration. The liquid is then dispensed into the corresponding AssayCup where the sample and other liquids were dispensed in the first pipetting step. The probe rises while dispensing the reaction mixture back into the AssayCup, thereby mixing the solution and accelerating the reaction in the AssayCup. The AssayTip is discarded into the solid waste tray when pipetting is complete.

Roche Diagnostics

cobas e 411

1 Mechanical theory

Assay sequence

Second incubation at 37 °C

If necessary, a second incubation step (4.5 or 9 minutes) occurs.

If using a pretreatment assay, the second incubation is similar to that described above for “First Incubation at 37 °C”.

Pipetting of additional reagent (pretreatment assays)

For pretreatment assays, reagent pipetting is similar to that described above for “Pipetting of additional reagent” occurs.

Third incubation at 37 °C (pretreatment assays)

If necessary, a third incubation step (9 minutes) occurs for pretreatment assays.

Aspirating the reaction mixture

In this process, the sipper probe first aspirates ProCell (tripropylamine solution, TPA) to prepare the measuring cell. Then, the sipper probe aspirates the reaction mixture from the AssayCup and transfers it to the measuring cell. The sipper probe is washed at the rinse station and ProCell is aspirated again to rinse away the unbound constituents of reagent and sample. Next, the ECL reaction in the measuring cell occurs.

Cleaning the measuring cell

Once the measurement is complete, the measuring cell is cleaned with CleanCell and prepared for a new measurement process.

It takes 42 seconds (one pipetting cycle) from the aspiration of the reaction mixture by the sipper probe until the measuring cell is filled with ProCell and ready for the next sample.

Finalization

Thirty minutes after documentation of the last result, the sipper pipetter flushes system water through the sipper probe, and then fills the measuring cell with ProCell before the analyzer returns to Standby mode.

After this procedure, every 30 minutes the waste pump of the S/R rinse station runs for 2 seconds (waste consumption approximately 12 mL). This procedure stops if the operation switch is switched off.

e Figure A-2 shows the finalization process for the cobas e 411 hardware.

Roche Diagnostics

1 Mechanical theory

cobas e 411

Assay sequence

Last sipping Ten cycles waiting for the new order Finalization Gripper moves to its home
Last sipping
Ten cycles waiting for the
new order
Finalization
Gripper moves to its home
position
Pipetter prime
Cleaning the sipper
flow with system water
Sipper
prime
Filling the measuring
Pipetter end wash
cell with
ProCell
Filling the sipper
nozzle with water

Standby

Figure A-2

Finalization process

Roche Diagnostics

cobas e 411

1 Mechanical theory

Workflow and throughput

Workflow and throughput

The workflow on the cobas e 411 analyzer system is entirely sample-orientated. Owing to the availability of a new disposable AssayTip for each test, there is no risk of contamination. Therefore it is possible to perform assays in any sequence, thus allowing samples to be completed one after the other.

When all assays on the system are 18-minute assays, the optimal throughput of 88 results per hour can be reached, producing a result every 42 seconds. In combination with 9- or 27-minute assays, or in combination with two-step dilution assays, the instrument will slow down, depending on the percentage and sequence of tests with other incubation times.

Effects of test combinations on throughput

The various available tests have different durations. The throughput of the cobas e 411 analyzer depends upon the way in which tests of a given duration are combined, as explained for each of the following combinations. There may be short periods of throughput slow-down on the disk system due to the loading of a new sample disk. Such gaps do not occur when the rack system is used, because the Roche Diagnostics/ Hitachi 5-position racks load continuously.

9-minute tests only

9-minute tests have two incubation periods, each of 4.5 minutes duration. If only 9- minute tests are performed, the optimal throughput will always be reached regardless of the test mixture.

All 9-minute tests follow the same time protocol. Therefore, there will be no timing conflicts. In one 42-second cycle, the cobas e 411 will simultaneously perform S1 (first reagent pipetting), S2 (second reagent pipetting), and D (detection).

18-minute tests only

18-minute tests have two incubation periods, each of 9 minutes duration. If only 18- minute tests are performed, the optimal throughput will always be reached regardless of the test mixture.

All 18-minute tests follow the same time protocol. Therefore, there will be no timing conflicts. In one 42-second cycle, the cobas e 411 will simultaneously perform S1 (first reagent pipetting), S2 (second reagent pipetting), and D (detection).

Combination of 9- and 18-minute tests

When tests of these two durations are combined, the throughput of the cobas e 411 depends on the percentage and distribution of the 9-minute tests. As a limiting factor, it is not possible to plan the detection of two tests during one 42 second cycle. When scheduling the first pipetting of a 9-minute test, the system has to be sure to have an open cycle for detection 9 minutes later. Depending on the percentage and distribution of the 9-minute assays, throughput may or may not be affected. If the number of requested 9-minute tests is very small, larger throughput gaps will exist.

Roche Diagnostics

1 Mechanical theory

cobas e 411

Workflow and throughput

27-minute tests only

27-minute tests have three incubation periods, each of 9 minutes duration. If only 27- minute assays are performed, the throughput of the cobas e 411 is reduced to 44 results per hour. Every 13 cycles, the cobas e 411 comes into a timing problem. It is not possible to perform a S0 (pretreatment pipetting) together with a S1 (first reagent pipetting) within one 42 second cycle. When this happens, the instrument will stand for 13 cycles (9 minutes) until it can pipette again without conflict.

Combination of 18- and 27-minute tests

When 18- and 27-minute assays are combined, the number of gaps created depends on the assay mix and on the exact test sequence. The gaps can vary from 1 to 13 idle cycles (42 seconds to 9 minutes). Limiting factors are that only one detection can take place during one 42-second cycle, and that S0 (the pretreatment step) cannot be combined with S1 (the first reagent pipetting).

Typical test durations

e Table A-2 contains details of the duration of some typical tests. This is not a complete list of tests, but is provided as an example.

Test

9 minutes

18 minutes

27 minutes

Thyroid

TSH, T3, FT3, T4, FT4, T-uptake, TG, Anti-TG, Anti-TPO

Fertility

hCG

LH, FSH, Prolactin, Prog, Testo, E2, HCG+beta, Cortisol, DHEAS, SHBG

Cardiac

CK-MB, Troponin T, Myoglobin

CK-MB, Troponin T, Myoglobin, Digoxin, Digitoxin, proBNP

Oncology

PSA, fPSA, CEA, AFP, CA 125m CA 15-3 II, CA 19-9, CA 72-4, Cyfra 21-1, NSE, S100

Infectious disease

HBsAg, anti-HBs, anti-HBc IgM (a) anti-HAV IgM * Anti-HAV, Anti, HBe, HBeAg, HIV Antigen, HIV combi

anti-HBc

Anemia

Ferritin

Vit B12, Folate

Diabetes

c-Peptide, e-Peptide, Insulin

Bone

B-CrossLaps, Total P1NP, N-MID Osteocalcin, PTH (b)

Other

IgE

Table A-2

Durations of some typical tests

(a)

18-minute test with two-step predilution

(b)

Under development

Roche Diagnostics

cobas e 411

1 Mechanical theory

Operation flow in analysis

Operation flow in analysis

e Figure A-3 shows a flow chart of the operational process.

Rerun

Assigned

Pre-start inspection

Pre-start inspection

Pre-start inspection
Pre-start inspection
the operational process. Rerun Assigned Pre-start inspection Switch on (Initialization and Standby) • Check alarm
Switch on (Initialization and Standby) • Check alarm button

Switch on (Initialization and Standby) • Check alarm button

Switch on (Initialization and Standby) • Check alarm button
Switch on (Initialization and Standby) • Check alarm button
on (Initialization and Standby) • Check alarm button Pre-routine operation Routine operation • Calibration and
Pre-routine operation

Pre-routine operation

Pre-routine operation
Pre-routine operation
and Standby) • Check alarm button Pre-routine operation Routine operation • Calibration and control • Routine
and Standby) • Check alarm button Pre-routine operation Routine operation • Calibration and control • Routine

Routine operation • Calibration and control

• Routine or STAT (a) sampling

------------------------------------------------------

Results

Results Sampling Stop (Finalization, Stop, and Standby) Maintenance
Sampling Stop (Finalization, Stop, and Standby)

Sampling Stop (Finalization, Stop, and Standby)

Sampling Stop (Finalization, Stop, and Standby)
Sampling Stop (Finalization, Stop, and Standby)
Results Sampling Stop (Finalization, Stop, and Standby) Maintenance Switch off ( a ) Short Turn Around
Maintenance

Maintenance

Maintenance
Maintenance
Sampling Stop (Finalization, Stop, and Standby) Maintenance Switch off ( a ) Short Turn Around Time

Switch off

(a) Short Turn Around Time

Figure A-3

Operational process

Roche Diagnostics

1 Mechanical theory

cobas e 411

Detailed assay sequence

Detailed assay sequence

The mechanical process of the instrument is described below with a sandwich test, TSH (thyroid-stimulating hormone), used as an example. This example assumes that the reagent pack was already registered by the analyzer and does not need calibration. All results are calculated on the basis of an existing lot calibration.

Preoperational steps

When Start is pressed from Standby mode, the following preoperational steps occur:

1. The analyzer resets all mechanisms to their respective home positions and accesses the data disk. Next, the S/R pipetter primes the S/R probe.

2. The gripper checks for an AssayTip in position number 1 of the AssayTip trays. If this position is empty, the gripper remembers where it last left off and checks that position. If this position is empty, the gripper considers the whole tray empty and the Inventory screen is updated accordingly.

tray empty and the Inventory screen is updated accordingly. If the analyzer is in S. Stop,

If the analyzer is in S. Stop, the gripper remembers where it last left off and checks for an AssayTip in that position.

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.

Roche Diagnostics

cobas e 411

1 Mechanical theory

Detailed assay sequence

Dispensation of reagent 1, reagent 2, and sample (disk system)

of reagent 1, reag ent 2, and sample (disk system) A TSH sample is present on

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.

rotates again to move the TSH reagent to the R1 position. A B A R1 aspiration

A

B

A R1 aspiration position

B

R2 aspiration position

Figure A-4

R1 and R2 aspiration positions

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.

Roche Diagnostics

1 Mechanical theory

cobas e 411

Detailed assay sequence

10. Upon completion of R2 aspiration, the S/R probe rises and moves to the rinse station. To prevent the aspirated R2 from coming into contact with the water in the rinse station, the probe aspirates another 10 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.

During sample aspiration, clot detection is activated. A A Sampling position Figure A-5 Sampling position (disk

A

A Sampling position

Figure A-5

Sampling position (disk system)

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.

Roche Diagnostics

cobas e 411

1 Mechanical theory

Detailed assay sequence

Dispensation of reagent 1, reagent 2, and sample (rack system)

of reagent 1, reage nt 2, and sample (rack system) A TSH sample is present on

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.

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.

e See Figure A-4 for the location of the R1 aspiration position.

While aspirating R1, the gripper puts another AssayTip in position 1 of the pipetting station.

7. If the S/R probe does not detect liquid during descent, no reagent aspiration can occur, an alarm is generated.

8. After R1 aspiration, the S/R probe rises and moves to the rinse station. To prevent the aspirated R1 from contacting the water in the rinse station, the probe aspirates 10 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.

e See Figure A-4 for the location of the R2 aspiration position.

11. Upon completion of R2 aspiration, the S/R probe rises and moves to the rinse station. To prevent the aspirated R2 from coming into contact with the water in the rinse station, the probe aspirates another 10 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

Roche Diagnostics

1 Mechanical theory

cobas e 411

Detailed assay sequence

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.

During sample aspiration, clot detection is activated. A Sampling position A Figure A-6 Sampling position (rack

A Sampling position

A

Figure A-6

Sampling position (rack system)

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.

Roche Diagnostics

cobas e 411

1 Mechanical theory

Detailed assay sequence

Microbead preparation

Before the first incubation is completed, the TSH microbeads are mixed to facilitate their aspiration and dispensation.

1.

The

reagent rotor rotates until the TSH reagent pack is at the reagent cap-open/

close mechanism. The mechanism moves out and opens the cap. The disk moves the reagent pack to the mixing position.

2.

The

mixer moves over the reagent rotor and descends into the microbeads to 1.4

mm

above the bottom of the bottle.

3.

The

mixer stirs the microbeads to obtain a homogeneous suspension.

4.

During the mixing, the gripper obtains a fresh AssayTip and transports it to position 2 of the pipetting station.

5.

When mixing is complete, the mixer rises and returns to the rinse station where it descends and rotates in the rinse station for washing.

6.

At the same time, the reagent rotor rotates the TSH reagent pack to the microbead pipetting position.

Microbead aspiration and dispensation

 

1.

The

gripper grasps the incubating cup and transports it to position 5 of the

 

pipetting station.

 

2.

The

S/R probe moves to the pipetting station and obtains the fresh AssayTip and

 

moves to the microbead pipetting position.

 

3.

While activating the liquid level detection, the S/R probe descends below the reagent surface and aspirates 40 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.

Roche Diagnostics

1 Mechanical theory

cobas e 411

Detailed assay sequence

Preparations for the measurement process

Before the second incubation is completed, the sipper probe aspirates ProCell into the measuring cell to facilitate measurement.

1. The sipper probe moves from its home position to a ProCell bottle and descends to 2 mm below the solution level and aspirates ProCell into the measuring cell. As the probe descends, liquid level detection is activated. The sipper probe can descend as low as 1.3 mm above the bottom of the ProCell bottle.

2. The sipper probe rises.

Measurement process

1. The gripper picks and transports the cup that has completed its second incubation from the incubator to the aspiration station.

2. The sipper probe moves to the aspiration station and descends into the cup.

3. When the sipper probe detects the reaction mixture in the cup, it aspirates 150 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.

Roche Diagnostics

cobas e 411

1 Mechanical theory

Dilution steps

Signal detection and conversion

The measuring cell is kept at a constant 28 °C throughout the measurement process. The photomultiplier tube detects and converts the ECL signal into an electric signal from which the cobas e 411 analyzer calculates assay results.

Automatic analyzer cycles

There are certain analyzer functions that occur automatically while the analyzer is switched on.

o

While the analyzer is in operation, the solid waste tray periodically shakes for 1.5 seconds.

o

While the analyzer is in Standby, the reagent rotor turns 90° every 30 minutes.

o

While the analyzer is in Standby, the rinse stations for the S/R probe and sipper probe are switched on for 2 seconds every 30 minutes.

o

Microbeads undergo a long mix when starting from Standby and then every 90 minutes until pipetting starts.

o

Microbeads undergo a short mix and then a short mix every 60 minutes for each reagent pack.

Dilution steps

The following is a description of how an assay with a dilution is performed, including the number of AssayTips and AssayCups used in the process.

Assay with one-step dilution

(1:2, 1:5, 1:10) AssayTip 1 ~ diluent (wash)* + sample

AssayTip 1

Diluent (wash)* + sample

AssayCup 1

AssayTip 2

R1 (wash)* + R2 (wash)*

AssayCup 2 (1st incubation)

AssayTip 3

M (wash)*

AssayCup 2 (2nd incubation)

Detection

Table A-3

* (wash) = the outside of the AssayTip is washed.

Dilution steps for an assay with one-step dilution (1:2, 1:5, 1:10)

R1

= Reagent 1

R2

= Reagent 2

M = Microbeads

Roche Diagnostics

1 Mechanical theory

cobas e 411

Pretreatment steps

Assay with two-step dilution

(1:50, 1:100)

AssayTip 1

Diluent (wash)* + sample

AssayCup 1

AssayTip 2

Diluent (wash)*

AssayCup 2

 

+

diluted sample from AssayCup 1

AssayTip 3

R1 (wash)* + R2 (wash)*

AssayCup 3

 

+

diluted sample from AssayCup 2

(1st incubation)

AssayTip 4

M (wash)*

AssayCup 3

 

(2nd incubation)

Detection

Pretreatment steps

Table A-4

* (wash) = the outside of the AssayTip is washed.

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

R1

= Reagent 1

R2

= Reagent 2

M = Microbeads

In certain test protocols, pretreatment reagent is added before R1, R2, or M, as summarized in the following table.

Pretreatment assay

AssayTip 1

PT1 (wash)* + PT2 (wash)*

AssayCup 1

 

+

sample

(1st incubation)

AssayTip 2

R1 + pretreated sample in AssayCup 1

AssayCup 1

 

(2nd incubation)

AssayTip 3

M (wash)* + R2

AssayCup 1

 

+

reaction mixture in AssayCup 1

(3rd incubation)

Detection

Table A-5

* (wash) = the outside of the AssayTip is washed. PT1 = Pretreatment 1 PT2 = Pretreatment 2

Pretreatment steps for an assay

R1

= Reagent 1

R2

= Reagent 2

M = Microbeads

Roche Diagnostics

cobas e 411

1 Mechanical theory

Analyzer status conditions

Analyzer status conditions

The cobas e 411 analyzer has a number of status conditions. The status conditions usually seen during routine operation or maintenance procedures are listed below. Several other status conditions, most them seen during various adjustment or maintenance procedures performed by a Roche Diagnostics service representative, are not included below.

e Refer to the Alarm screen for further information about instrument alarms.

A. Stop (analyzer stop)

information about instrument alarms. A. Stop (analyzer stop) The analyzer is no longer able to continue

The analyzer is no longer able to continue operation. An alarm was issued. Take the appropriate measures to resolve the problem.

A. Stop/L. Stop (analyzer stop/line stop)

The analyzer is already in A. Stop status when the lines stop operation. the problem. A. Stop/L. Stop (ana lyzer stop/line stop) A. Stop/R. Stop (analyzer stop/rack stop) The

A. Stop/R. Stop (analyzer stop/rack stop)

stop operation. A. Stop/R. Stop (analyzer stop/rack stop) The analyzer is already in A. Stop status

The analyzer is already in A. Stop status when the A-Line stops supplying racks to the B-Line.

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.

Roche Diagnostics

1 Mechanical theory

cobas e 411

Analyzer status conditions

Finalization maint.

This status occurs when Finalization Maintenance is initiated from the Maintenance screen.

Initialization

This status is seen when the cobas e 411 analyzer is switched on, or when Start is pressed from Standby.

L.

& A. reset all (line & analyzer)

L. and A. reset all status occurs when the corresponding function is initiated from the

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.

L.

Stop (line stop)

All lines stop operation. An alarm was issued. Take the appropriate measures to resolve the problem.the analyzer and the lines. L. Stop (line stop) Liquid flow cleaning Liquid flow cleaning occurs

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.

Roche Diagnostics

cobas e 411

1 Mechanical theory

Analyzer status conditions

R. Stop (rack stop)

theory Analyzer status conditions R. Stop (rack stop) This status occurs when there are no more

This status occurs when there are no more racks to process on the A-Line or B-Line.

Rack clear

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. Maintenance screen. This function clears any remaining racks on the A-, B- or C- Lines.

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)

This status occurs when S. Stop is pressed or when sampling is complete.

This status occurs when S. Stop is pressed or when sampling is complete.

S.

Stop-S. Scan

pressed or when sampling is complete. S. Stop-S. Scan Sample scan The analyzer is in S.

Sample scan

sampling is complete. S. Stop-S. Scan Sample scan The analyzer is in S. Stop and a

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.

Roche Diagnostics

1 Mechanical theory

cobas e 411

Analyzer status conditions

Sipper LLD volt.

The analyzer is monitoring the liquid level detection voltage of the sipper probe. The check is initiated from the Voltage Monitor screen (Utility) folder.

Sipper pipet. prime

This status occurs when the sipper pipetter prime is initiated from the Maintenance screen.

Standby

 

The analyzer is not performing any operations.

Stop

 

This status occurs when Stop is pressed or when a Stop alarm condition exists. If an alarm exists, take the appropriate measures to resolve the problem.

System reset

A system reset is initiated from the Maintenance screen.

Roche Diagnostics

Measurement technology

B

2 ECL technology

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

B-3

cobas e 411

2 ECL technology

Table of contents

ECL technology

This chapter provides an overview of the electrochemiluminescent technology in the cobas e 411 analyzer system. The use of the ruthenium complex and the measuring cell in which the reaction occurs are described.

In this chapter

Chapter

2

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

Roche Diagnostics

2 ECL technology

cobas e 411

Table of contents

Roche Diagnostics

cobas e 411

2 ECL technology

ECL measuring principles

ECL measuring principles

Electrochemiluminescent (ECL) processes are known to occur with numerous molecules, including compounds of ruthenium, osmium, rhenium, and other elements.

ECL is a process in which highly reactive species are generated from stable precursors at the surface of an electrode. These highly reactive species react with one another, producing light.

The development of ECL/Origen immunoassays is based on the use of a

complex and tripropylamine (TPA). The

2+

ruthenium(II)-tris(bipyridyl) [Ru(bpy) 3 ]

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.

technology to be applied to a wide variety of analytes. 2 + O O N N
2 + O O N N N N O O Ru N N N Figure
2 +
O
O
N
N
N
N
O
O
Ru
N
N
N
Figure B-1
The ruthenium complex

Roche Diagnostics

2 ECL technology

cobas e 411

ECL measuring principles

The ECL reaction at the electrode surface

Photon Diffusion Magnetic TPA• microbead -H + TPA TPA• + e - e - Electrode
Photon
Diffusion
Magnetic
TPA•
microbead
-H +
TPA
TPA• +
e -
e -
Electrode
Figure B-2
Detection of a ruthenium-labeled immune complex

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.

Roche Diagnostics

cobas e 411

2 ECL technology

ECL measuring principles Electrode surface TPA• + - e -H + TPA TPA• - e
ECL measuring principles
Electrode
surface
TPA• +
-
e
-H +
TPA
TPA•
-
e
Ru(bpy) 3 3+
-
e
Ru(bpy) 3 2+
2+
Ru(bpy)
excited
ground
3 state
Photon
state
(620 nm)

Figure B-3

The ECL reaction at the electrode surface

TPAo and Ru(bpy) 3 3+ react with one another, whereby Ru(bpy) 3 3+ is reduced to

2+

Ru(bpy) 3

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.

and at the same time forms an excited state through energy transfer. This

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.

Roche Diagnostics

2 ECL technology

cobas e 411

ECL measuring principles

ECL signal generation

The following figure illustrates a typical ECL signal generation. Viewed from an electrical perspective, the reaction can be explained as follows: When a voltage is applied to the electrode of the measuring cell, a brief peak of light emission occurs, which can be detected as the resulting ECL signal. A defined area under the curve is measured around the intensity maximum.

ECL intensity (counts) applied voltage [mV] 1500 350,000 1200 300,000 900 250,000 200,000 600 150,000
ECL intensity (counts)
applied voltage [mV]
1500
350,000
1200
300,000
900
250,000
200,000
600
150,000
300
100,000
50,000
0
0
0.00
0.20
0.40
0.60
0.80
1.00
1.20
Time [s]
Figure B-4
ECL signal generation

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.

Roche Diagnostics

cobas e 411

2 ECL technology

ECL measuring principles

ECL measuring cell

The core of the detection unit is the ECL measuring cell, which is designed as a flow- through cell. The following figure shows the main components of the measuring cell:

A

B

C

D

E

F G H I J O N M L K A Screw B Counter electrode
F
G
H
I
J
O
N
M
L
K
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

 

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

Roche Diagnostics

2 ECL technology

cobas e 411

Advantages of ECL technology

photomultiplier. The system then uses the corresponding signals for the calculation of results.

o Release of microbeads and cell cleaning

Once the measurement is completed, the paramagnetic microbeads are washed away from the electrode surface with a special cleaning solution (CleanCell). The surface of the measuring cell is regenerated by varying the potential on the electrode. The cell is then ready for another measurement.

B C

A D F
A
D
F

G

E

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

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.

Roche Diagnostics

Test principles

C

3 Test principles

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

C-3

4 Reagent concept

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

C-13

cobas e 411

3 Test principles

Table of contents

Test principles

This chapter provides an overview of the immunology test principles used by the cobas e 411 analyzer.

In this chapter

Chapter

3

Test principles

5

Competitive principle

6

Sandwich principle

8

Bridging principle

10

Roche Diagnostics

3 Test principles

cobas e 411

Table of contents

Roche Diagnostics

cobas e 411

3 Test principles

Test principles

Test principles

Three test principles are available on the cobas e 411 analyzer:

o Competitive principle for extremely small analytes o Sandwich principle (one or two steps) for
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:
Sandwich principle for high
molecular weight analysis
Bridging principle to
determine IgG and IgM
Competitive principle for low
molecular weight haptens
Analyte
ECL label
Antibody
Surface of para-
magnetic microbead
Streptavidin-biotin
binding
Figure C-1
ECL assay 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

Roche Diagnostics

3 Test principles

cobas e 411

Test principles

Competitive principle

This principle is applied to analytes of low molecular weight, such as free triiodothyronine (FT3).

e

Refer to Figure C-2 on page C-7 for an illustration of the competitive principle.

o

In the first step, sample and a specific anti-T3 antibody labeled with a ruthenium complex are combined in an assay cup.

o

After the first incubation, biotinylated T3 and streptavidin-coated paramagnetic microbeads are added. The still-free binding sites of the labeled antibody become occupied, with formation of an antibody-hapten complex. The entire complex is bound to the microbeads through the interaction of biotin and streptavidin.

o

After the second incubation, the reaction mixture containing the immune complexes is transported into the measuring cell. The immune complexes are magnetically captured on the working electrode, but unbound reagent and sample are washed away by ProCell.

o

In the ECL reaction, the conjugate is a ruthenium-based derivative and the chemiluminescent reaction is electrically stimulated to produce light. The amount of light produced is indirectly proportional to the amount of antigen in the patient sample.

The concentration of the antigen is evaluated and calculated by means of a calibration curve that was established using standards of known antigen concentration.

Roche Diagnostics

cobas e 411

3 Test principles

Test principles