cobas c 311_cobi_en

cobas c 311 analyzerCOBI CD Compendium of Background Information

cobas c 311 analyzer

Roche Diagnostics

2 COBI CD Version 1.0

Document information

Revision history

Edition notice cobas c 311 analyzer Compendium of Background Information

This document is for users of the cobas c 311 analyzer.

Every effort has been made to ensure that all the information contained in this manual is correct at the time of printing. However, Roche Diagnostics GmbH reserves the right to make any changes necessary without notice as part of ongoing product development.

Any customer modification to the instrument will render the warranty or service agreement null and void.

Software updates may only be carried out by Roche Service representatives.

Intended use This document is intended to provide background information for a better understanding of the hardware, test principles and calibration methods of the cobas c 311 analyzer.

Copyright 2007, Roche Diagnostics GmbH. All rights reserved.

Trademarks The following trademarks are acknowledged:

COBAS, COBAS C, and LIFE NEEDS ANSWERS are trademarks of Roche.

All other trademarks are the property of their respective owners.

Instrument approvals The cobas c 311 analyzer meets the protection requirements laid down in IVD Directive 98/79/EC. Furthermore, our instruments are manufactured and tested according to the following international standards:

o IEC 61010-1: 2001

o IEC 61010-2-010: 2003

o IEC 61010-2-081: 2001

o IEC 61010-2-101: 2002

o UL 61010-1: 2001

o CAN/CSA C22.2 No. 61010-1-04

o EN 61326-2-6:2006

Compliance is demonstrated by the following marks:

COBI CD version

Edition for Revision date Changes

1.0


April 2007

Complies with the IVD directive 98/79/EC.

C US

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

US.

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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


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Table of contents

Document information 2Contact addresses 3Table of contents 5How to use the CD 7Installation of Adobe Acrobat Reader 7Where to find information 7Online Help system 8

Measurement technology Part A

1 Photometric technologyGeneral photometer characteristics A-5

Test principles Part B

2 ISE unit - Ion selective electrode principlesIntroduction B-5Calculation of unknown sample concentrations B-5

3 Photometric principlesTypes of photometric assays B-9Comprehensive assay descriptions B-12Reaction cell and calibration data B-21Endpoint assays B-24Rate assays B-30Prozone check B-39Summary of assay techniques B-44

4 Serum index principlesIntroduction B-49Definition of serum indices B-49Measurement of serum indices B-49Evaluating serum indices B-51Serum index data alarms B-51

Calibration Part C

5 ISE unit - Ion selective electrode calibrationISE calibration C-5Slope calculation C-6Internal standard calculation C-6One-point calibration C-7Compensation overview C-7Compensation value calculation C-7Reference electrode C-8

6 Photometric calibrationCalibration checks C-11Calibration overview C-14Linear calibration C-22RCM calibration C-25RCM2T1 calibration C-27RCM2T2 calibration C-29Spline calibration C-31Line Graph calibration C-33

Calculating data alarms Part D

7 Calculating data alarmsIntroduction D-5Prozone effect D-5Linearity verification (>Lin) D-7Sensitivity limit check (Sens.E) D-9Duplicate limit check (Dup.E) D-9Technical limit check (>Test) D-11Repeat limit check (>Rept) D-12Reaction limit check (>React) D-12

Quality control Part E

8 Applying QC rulesIntroduction E-5Rule 1: 1-2SD E-6Rule 2: 1-2.5SD (Q2.5SD alarm) E-6Rule 3: 1-3SD (Q3SD alarm) E-7Rule 4: 2-2SA (S2-2Sa alarm) E-8Rule 5: R-4SD (R4SD alarm) E-9Rule 6: 2-2SW (S2-2Sw alarm) E-10Rule 7: 4-1SA (S4-1Sa alarm) E-11Rule 8: 4-1SW (S4-1Sw alarm) E-12Rule 9: 10XA (S10Xa alarm) E-13Rule 10: 10XW (S10Xw alarm) E-14

Index Part F

Index F-3

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How to use the CD

This CD is provided as an information source for background knowledge regarding the cobas c 311 analyzer. Some of the information on this CD is available in PDF-format and requires Adobe Acrobat Reader to be installed. If you do not have this software installed, refer to the instructions for the Installation of Adobe Acrobat Reader below. You may access information by selecting a topic from the table of contents on the left.

If you have any further questions, please do not hesitate to contact Roche Diagnostics Customer Service or visit us on the Web at www.roche.com/diagnostics.

Installation of Adobe Acrobat Reader

We have included the files necessary to install Adobe Acrobat Reader in this CD. If this software is not installed on your computer, proceed as follows:

1 Close all running applications.

2 Change to the folder \reader on the CD-ROM.

3 Double-click on AdbeRdr707_en_US.exe to start the installation routine for Adobe Acrobat Reader.

4 Follow the instructions on screen.

5 It is recommended that you restart your computer after the installation process has finished.

Where to find information

The following documents are provided to assist in finding desired information quickly:

Operators Manual Contains information about safety, hardware components and operating the analyzer as well as maintenance and troubleshooting. A table of contents at the beginning of the manual as well as at the beginning of each chapter, and an index at the end of this manual help you to find information quickly.

Online Help Contains a detailed description of the software of the cobas c 311 analyzer. In addition to the software description, the whole Operators Manual is included in the Online Help. This makes it possible to retrieve information from both Online Help and Operators Manual using the search functions available for electronically stored documents.

COBI CD The COBI CD (Compendium of Background Information) provides you with background information about the technologies, test principles, their theory and calibration methods used by the cobas c 311 analyzer. It also provides a complete glossary. The information can be read and printed using Adobe Acrobat Reader.


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Online Help system

The software of the cobas c 311 analyzer has a context sensitive Online Help feature to aid you in operating the instrument. "Context sensitive" means that wherever you are located within the cobas c 311 software, choosing the Help feature displays Help text or a screenshot relating to that area of the software. The Online Help offers a quick and convenient way to find information, such as explanations of screens and dialog boxes and how to perform particular processes.

F1 Help There are two ways to enter the Online Help: via the Help icon in the bottom left of the screen or by pressing F1 on the keyboard. The context sensitive entry displays information relating to your current location in the software.

1 Photometric technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-3

Measurement technology A

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cobas c 311 analyzer 1 Photometric technologyTable of contents

Photometric technology

This chapter provides you with an overview of the application of photometric technology in the cobas c 311 analyzer.

General photometer characteristics .......................................................................... A-5

In this chapter Chapter 1

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A-4 COBI CD Version 1.0

1 Photometric technology cobas c 311 analyzerTable of contents

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COBI CD Version 1.0 A-5

cobas c 311 analyzer 1 Photometric technologyGeneral photometer characteristics

General photometer characteristics

An illustration of the light path is shown below.

When the light beam enters the photometer, it strikes a diffraction grating, which separates the light into its constituent wavelengths and reflects them onto a fixed array of 12 photodiodes. Each photodiode is permanently positioned to detect light at a different wavelength.

Absorbance readings are taken each time a reaction cell rotates past the photometer. When the reaction cell passes through the photometer lightpath, absorbance at the 12 wavelengths for each individual assay is measured.

Most Roche Diagnostics photometric tests use two wavelength readings to calculate results. The end product of a chemical reaction absorbs the most light at one particular wavelength. However, using the difference between readings at two wavelengths (bichromatic system) eliminates the effect of interferences sometimes found when using a single wavelength (monochromatic system) and compensates for most of the photometric noise which improves the photometric resolutions.

For each reaction cell, a waterblank is measured and then absorbance readings are taken 57 times (57 measure points) in 10 minutes.

Choice of wavelengths Bichromatic analysis uses two wavelengths: One that is at or near the peak absorbance of the chromogen produced by the reaction, and a second wavelength at which little or no absorbance of the desired chromogen occurs.

A GratingB PhotometerC SlitD Imaging lensE Slit (out)

F Reaction cell and contentsG Incubator bathH Slit (in)I Condenser lensJ Mask

K Infrared cut filterL Water jacket M Photometer lampN Detector

Figure A-1 Photometer lightpath

C EA D F G H I JB K L M

N

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A-6 COBI CD Version 1.0

1 Photometric technology cobas c 311 analyzerGeneral photometer characteristics

Any absorbance ( ) that occurs, due to interference from other substances in the sample, is measured at the secondary wavelength. This amount is then subtracted from the total absorbance ( ) occurring at the primary wavelength to yield the net absorbance ( ).

The optimum measure points for each test are part of the application parameters, which are available via download.

The assay code and calibration type programmed from the application parameters determine how final results are calculated for each test.

Figure A-2 Bichromatic absorbance

A2

A1AC

Abs

orba

nce

Wavelength

Chromophore

Observed

AC

A2

1 2

Interferent

A1

2 ISE unit - Ion selective electrode principles . . . . . . . . . . . . . . . . B-3

3 Photometric principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-7

4 Serum index principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-47

Test principles B

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COBI CD Version 1.0 B-3

cobas c 311 analyzer 2 ISE unit - Ion selective electrode principlesTable of contents

ISE unit - Ion selective electrode principles

This chapter provides you with an overview of the ion selective electrode test principles and result calculation used by the cobas c 311 analyzer.

Introduction ............................................................................................................... B-5

Calculation of unknown sample concentrations ...................................................... B-5


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B-4 COBI CD Version 1.0

2 ISE unit - Ion selective electrode principles cobas c 311 analyzerTable of contents

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cobas c 311 analyzer 2 ISE unit - Ion selective electrode principlesIntroduction

Introduction

The ISE unit performs indirect measurement of electromotive force (EMF) in millivolts between ion selective electrodes and the reference electrode. Indirect measurement means that all samples are diluted at a 1:31 ratio.

The EMF values of each sample are converted to mmol/L values by a calculation algorithm that uses the EMF data together with data from a two-point calibration with two primary standards.

A one-point calibration before and after each routine sample measurement is used to offset the drift between consecutive measurements. For this one-point calibration the internal standard (IS) is used.

Calculation of unknown sample concentrations

The concentration of the sodium, potassium, and chloride in a sample is calculated from the EMF of the specific electrode by the following equation, which is derived from the Nernst Equation:

Equation B-1

Concentration of the specific ion in sample

Compensation value

Concentration of the internal standard

Electromotive force (voltage) of the unknown sample for the specific ion

Electromotive force (voltage) of the internal standard for the specific ion

Slope

Cs C.Value CIS 10 Es EIS( ) S+=

Cs

C.Value

CIS

Es

EIS

S

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2 ISE unit - Ion selective electrode principles cobas c 311 analyzerCalculation of unknown sample concentrations

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3 Photometric principles cobas c 311 analyzerTable of contents

Photometric principles

This chapter provides you with an overview of the photometric test principles and assay techniques used by the cobas c 311 analyzer.

Types of photometric assays ...................................................................................... B-9

Assay types and measure points .......................................................................... B-9

Displaying assay type and measure points ........................................................ B-11

Comprehensive assay descriptions .......................................................................... B-12

Example of a 2 Point End assay ......................................................................... B-12

Example of a Rate A assay .................................................................................. B-17

Reaction cell and calibration data ........................................................................... B-21

Cell Blank Measurement report ......................................................................... B-21

Working Information window .......................................................................... B-22

Others tab ........................................................................................................... B-23

Endpoint assays ........................................................................................................ B-24

1 Point assay ........................................................................................................ B-24

1 Point assay graph ....................................................................................... B-25

Sample program and calculations ............................................................... B-26

2 Point End assay ................................................................................................ B-27

2 Point End assay graph ............................................................................... B-27


Rate assays ................................................................................................................. B-30

Rate A assay ......................................................................................................... B-30

Rate A assay graph ........................................................................................ B-30


Rate A assay with sample blank correction ....................................................... B-33

Rate A assay with sample blank graph ........................................................ B-33


2 Point Rate assay ............................................................................................... B-36

2 Point Rate assay graph - R1 and R2 or R3 timing .................................... B-36


Prozone check ........................................................................................................... B-39


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3 Photometric principles cobas c 311 analyzer

Antigen readdition method ............................................................................... B-39

Programming and calculation ..................................................................... B-40

Calculation example ..................................................................................... B-41

Reaction rate method ......................................................................................... B-42

Programming and calculation ..................................................................... B-43

Summary of assay techniques .................................................................................. B-44

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cobas c 311 analyzer 3 Photometric principlesTypes of photometric assays

Types of photometric assays

There are two fundamental types of photometric assays on this instrument:

o Endpoint assays

o Rate assays

Measurements are taken by the photometer at specific measure points. If measurements are taken after the reactions are completed, the intensity of the colored (or turbidity) product is an indicator of the sample component's concentration. These are called endpoint assays.

For rate assays, the rate of the reaction is proportional to the concentration or activity of the sample component being analyzed. Measurements are taken as the reaction proceeds.

There are also modifications of these two techniques possible in this instrument, as well as a combination of the two.

Assay types and measure points

There are four different assay types. The assay types are divided in endpoint assays and rate assays:

e For more information on endpoint assays, see:1 Point assay on page B-24

2 Point End assay on page B-27

e For more information on rate assays, see:Rate A assay on page B-30

Rate A assay with sample blank correction on page B-33

2 Point Rate assay on page B-36

Fundamental assay type Assay type Characteristic

Endpoint assays 1 Point Endpoint assay programmed for a single measure

point

2 Point End Endpoint assay with sample blank

Rate assays Rate A Rate assay applying least squares method on

multiple measure points

2 Point Rate Rate assay programmed for two measure points

Table B-1 Assay types

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3 Photometric principles cobas c 311 analyzerTypes of photometric assays

Measure points Independent of the programmed application parameters, the photometer measures the absorbance of a reaction mixture in fixed intervals of 3 to 24 seconds. Not all of these measurements are used for the calculation of the result. Therefore, the numbering of the photometer measure points differs form the numbering of the measure points used in calculations.

The figure below represents an endpoint assay programmed for two measure points ( and ).

In this example, the application parameters define the 6th photometer measure point ( ) to be and the 24th photometer measure point ( ) to be . In other words, of the instrument is set to be of the test calculation, and of the instrument is set to be of the test calculation.

Figure B-1 Photometer measure points

mp1 mp2

mp6 mp1 mp24 mp2mp6 mp1 mp24

mp2

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cobas c 311 analyzer 3 Photometric principlesTypes of photometric assays

Displaying assay type and measure points

The Analyze tab on the Utility > Application screen displays the assay type and measure points among other application parameters for a selected test.

a To view the assay type and measure points for a test

1 Select Utility > Application.

2 Select the test you want to view from the test list on the left side of the screen.

3 Select the Analyze tab.

4 To the right of Assay/Time/Point there are six text boxes:

o The first entry displays the assay type selected.

o The second entry displays the reaction time in minutes.

o The third through sixth entries display chosen measure points.

In the following sections, the entries for the Assay/Time/Point text boxes on Utility > Application > Analyze are shown as follows:

Assay/Time/Point: [ Assay Type ] [ time ] [ ] [ ] [ ] [ ]

Figure B-2 Analyze tab on Utility > Application screen

mp1 mp2 mp3 mp4

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3 Photometric principles cobas c 311 analyzerComprehensive assay descriptions

Comprehensive assay descriptions

In the following section one example of an endpoint assay and one example of a rate assay is given, along with detailed explanations of the application parameters and result calculations.

e For extended programming and calculation examples, see:Example of a 2 Point End assay on page B-12

Example of a Rate A assay on page B-17

Example of a 2 Point End assay

A 2 Point End assay is an endpoint assay with sample blank measurement and can be programmed for two or more reagents. 2 Point means there are readings at two measure points, and :

o is the sample blank which is measured before or shortly after the final reagent is added.

o measures the absorbance of the final reaction product; it is set after addition of final reagent and after the reaction is completed.

2 Point End assay graph A graphic representation of a 2 Point End assay using reagents dispensed at R1 and R2 timing is shown below.

mp1 mp2

mp1

mp2

Figure B-3 2 Point End assay graph

C1, C2, ... The reaction cell's water blank values(a)

(a) See Cell Blank Measurement report on page B-21.

Pipetting of sample

Pipetting of reagent at R1 timing


1st photometric measure point (sample blank)

2nd photometric measure point (endpoint)

, Absorbances at measure point 1 and measure point 2

Abs

orba

nce

Time

S

C1 C2 C3

mp2

R2

mp1

R1

Amp1

Amp2

S

R1

R2

mp1

mp2

Amp1 Amp2

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cobas c 311 analyzer 3 Photometric principlesComprehensive assay descriptions

Example data The following data from the Utility > Application screen are used for this example:

Entries on Utility >Application > Analyze

In the later sections, the entries for the Assay/Time/Point text boxes on Utility > Application > Analyze are shown as follows:

Assay/Time/Point: [ 2 Point End ] [ 10 ] [ 6 ] [ 24 ] [ 0 ] [ 0 ]

This means:

o The assay type is 2 Point End.

o The reaction time is 10 minutes.

o The sample blank absorbance (sample plus first reagent) is determined by the 6th photometer measurement of the respective reaction cell.

o The absorbance of the sample plus first and second reagents is determined by the 24th photometer measurement of the respective reaction cell.

Dilution factor After the mixture of sample and R1 reagent is measured as sample blank, it is diluted by the addition of R2 reagent. Therefore, the readings cannot be subtracted, unless a correction for the dilution is taken into account. A dilution factor ( ) is calculated as follows and applied to the sample + R1 absorbance:

Equation B-2

Test GLUC2

Assay type 2 Point End

Time 10 min

Points 6, 24

2nd wavelength 700 nm

Primary wavelength 340 nm

Conc. value for Std (1) 0.0

Figure B-4 Entries on Utility > Application > Analyze

Dilution factor

Sample volume

R1 volume

R2 volume

d

dVsamp VR1+

Vsamp VR1 VR2+ +----------------------------------------------=

d 2L 150L+2L 150L 50L+ +----------------------------------------------------

152202--------- 0,7525= = =

d

Vsamp

VR1

VR2

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Reaction monitor The two measure points for this assays calculation are set at the 6th and 24th photometer measurements; the first is the sample blank reading, the second is the final absorbance reading (endpoint), as indicated in the Reaction Monitor below.

You can move the focus from one measure point to the next using the scroll bar below the graph. The absorbance at the measure point that has the focus is displayed in the Abs. field above the graph. Alternatively, the absorbance values of all measure points are listed on the Reaction Monitor report also:

The values on the Reaction Monitor report (as well as those in the Abs. field on the Reaction Monitor window) are absorbance 104. Moreover, these values are already corrected for the water blank value, determined during the cell blank measurement.

e See Cell Blank Measurement report on page B-21.

The real time water blank values displayed in the CB1-3 column of the Reaction Monitor report serve to verify the integrity of the reaction cell immediately before sampling.

Figure B-5 Reaction Monitor window of a 2 Point End assay

Reaction Monitor 06/02/07 17:54

Ser/Pl N000001 001 11/01/07 CELL 055 GLUC2 5.3ID 13:53:33

*** (PRIMARY) - (SECONDARY) *** CB1-3 01-10 11-20 21-30 31-40 41-50 51-573239 1864 4601 4611 4606 4608 46113240 1819 4609 4610 4609 4609 46133240 1773 4607 4608 4611 4612 4610

1765 4608 4607 4605 4609 46141759 4611 4610 4610 4609 46121750 4607 4604 4610 4610 46082102 4603 4608 4608 4609 46073963 4610 4608 4612 46124474 4608 4607 4604 46094576 4609 4608 4607 4610

Figure B-6 Reaction Monitor report

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Reaction absorbance To determine the reaction absorbance , the sample blank value is corrected for dilution and then subtracted from the endpoint absorbance:

Equation B-3

The absorbance used in calculations ( ) is 0.3290.

Calculation of concentration The calculation of the unknown concentration of the analyte in the sample uses the following endpoint reaction formula:

Equation B-4

and are displayed on the Working Information window. Select Calibration > Status > Calibration Result > Working Information to display this window.

When the test's concentration value for Std (1) is programmed with a decimal, the displayed K factor includes an extra digit for each number to the right of the decimal point.

is the absorbance of the first standard solution, Std (1), which is a blank calibrator. This value is also displayed on the Working Information window in the S1 Abs. field.

e See Working Information window on page B-22.

Ax

Ax Amp24 d Amp6=Ax 0,4607 0,7525 0,1750=Ax 0,4607 0,1317 0,3290= =

Ax

Concentration of the analyte (Gluc) in the sample

Calibration factor (also referred to as K factor)

Absorbance after reaction is completed (calculated above: 0.3290)

Absorbance of Std (1)/blank calibrator (S1 Abs.)

Concentration value for Std (1)/blank calibrator

, Instrument constants representing a slope of 1 and an intercept of 0

Figure B-7 Working Information window

Cx K Ax Ab( ) Cb+[ ] IFA IFB+=

Cx

K

Ax

Ab

Cb

IFA IFB

K Ab

Ab

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, the concentration of the analyte in the first standard solution Std (1), is displayed on the Others tab of the Utility > Application screen. This value controls the number of digits of the displayed and the rounding of the final results. When the test's value is programmed with a decimal, includes an extra digit for each number to the right of the decimal point.

e See Others tab on page B-23.

Example values The following values are used for this example:

Applying these values to the above formula yields:

The result is rounded to 5.3 on the report because , the concentration value for Std (1), the blank calibrator, contains one zero to the right of the decimal point as displayed on Utility > Application > Others.

CbCb

KCb K

16.3 (displayed as 163 due to a Std (1) concentration value of 0.0)

0.3290 (calculated above)

0.0030 (displayed as 30 in the S1 Abs. field due to factor 104)

0.0

, Instrument constants representing a slope of 1 and an intercept of 0

K

Ax

Ab

Cb

IFA IFB

Cx K Ax Ab( ) Cb+[ ] IFA IFB+=Cx 16,3 0,3290 0,0030( ) 0,0+=Cx 16,3 0,3260( )=Cx 5,314=

Cb

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Example of a Rate A assay

For rate assays, the time course of the reaction is followed by measuring the absorbance as a function of time. That is, measurements are taken as the reaction proceeds. Rate assays use these measurements because their concentration calculations are based on the determination of the rate of change in absorbance, :

Equation B-5

A Rate A assay is programmed for multiple measure points. This means, there is a measuring window and every photometric measurement within this window is taken into account for the rate calculationbeginning with the reading at the first programmed measure point ( ) through the reading at the second programmed measure point ( ).

The absorbance values are converted into the rate of change in absorbance ( ) by least squares analysis. There is no need for a dilution factor because all readings are taken after the addition of the last reagent.

Rate A assay graph A graphic representation of a Rate A assay using a reagent dispensed at R1 and R2 or R3 timing is shown below.

v

vxdAxdt

---------=

mpinitialmpfinal

v

Figure B-8 Rate A assay - reagents at R1 and R2 or R3 timing

C1, C2, ... The reaction cell's water blank values(a)


S Pipetting of sample

R1 Pipetting of reagent at R1 timing

R2/R3 Pipetting of reagent at R2/R3 timing

Rate of change in absorbance (slope) between and

First photometric measure point

Last photometric measure point

Abs

orba

nce

S, R1

C1 C2 C3

mp1 Timemp2

Absorbance limit

Blank

R2/R3

vx

vx mp1 mp2

mp1

mp2

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Example data The following data from Utility > Application screen are used for this example:


In the later sections of this chapter, the entries for the Assay/Time/Point text boxes on Utility > Application > Analyze are shown as follows:

Assay/Time/Point: [ Rate A ] [ 10 ] [ 12 ] [ 31 ] [ 0 ] [ 0 ]

This means:

o The assay type is Rate A.

o The reaction time is 10 minutes.

o The initial absorbance reading is the 12th photometer measurement of the respective reaction cell.

o The final absorbance reading is the 31st photometer measurement of the respective reaction cell.

Reaction monitor The rate of change in absorbance is calculated by least squares analysis of the absorbance values measured within the measuring window, as indicated in the reaction monitor below:

The values on the reaction monitor report are reaction absorbance 104. Moreover, these values are already corrected for the water blank value determined during the cell blank measurement.

e See Cell Blank Measurement report on page B-21.

Test ALTL

Assay Rate A

Time 10 min

Points 12, 31

2nd wavelength 700 nm

Primary wavelength 340 nm

Conc. value for Std (1) 0.00

Figure B-9 Entries on Utility > Application > Analyze

Figure B-10 Reaction Monitor window of a Rate A assay

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The absorbance values measured between the initial and the final absorbance reading ( through ) represent a change over 4.05 minutes. The mathematical analysis results in a rate of change in absorbance of -0.0503 per minute.

Result calculation The calculation of the unknown concentration of the analyte in the sample uses the following rate reaction formula:

Equation B-6

and are displayed on the Working Information window. Select Calibration > Status > Calibration Result > Working Information to display this window.

mp12 mp31


Ser/Pl N000001 063 16/02/07 CELL 58 ALTL 2.11ID 14:30:43

*** (PRIMARY)-(SECONDARY) ***CB1-3 1-10 11-20 21-30 31-40 41-50 51-573465 3505 19230 18231 17102 16511 159903467 3490 19130 18126 17008 16477 158853466 3489 19032 18026 16908 16410 15794

3486 18932 17933 16872 16377 15690 3478 18837 17822 16809 16303 15584 3477 18729 17610 16772 16278 1549519560 18638 17502 16703 16214 1539319503 18535 17396 16674 1617919420 18428 17306 16609 1611719328 18328 17201 16575 16084


Calibration factor

Rate of change in absorbance (expressed in 104/min)

Rate of change in absorbance of the reaction with Std (1)/blank calibrator


Concentration of the analyte (ALT) in the sample

, Instrument constants for a slope of 1 and an intercept of 0

Cx K vx vb( ) Cb+[ ] IFA IFB+=

K

vx

vb

Cb

Cx

IFA IFB

K vb

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When the test's concentration value for Std (1) is programmed with a decimal, the displayed K factor includes extra digits for each number to the right of the decimal point. is displayed in the S1 Abs. field of the Working Information window.

e See Working Information window on page B-22.

, the concentration of the analyte in the first standard solution, Std (1), is displayed on the Others tab of the Utility > Application screen.

e See Others tab on page B-23.

Example values The following values are used for this example:

Applying these values to the rate reaction formula (Equation B-6) yields:

The result is displayed as 2.11 on the report because , the concentration value for Std (1), the blank calibrator, contains two zeroes to the right of the decimal point as displayed on Utility > Application > Others.


vb

Cb

-42.04 (displayed as -4204 due to a Std (1) concentration value of 0.00)

-0.0503/min (calculated by least squares method)

-0.0001/min (displayed as -1 in the S1 Abs. field due to factor 104)

0.00

, Instrument constants for a slope of 1 and an intercept of 0

K

vx

vb

Cb

IFA IFB

Cx 42,04 0,0503 0,0001( )[ ] 0,0+{ } 1 0+=Cx 42,04 0,0502( )=Cx 2,110=

Cb

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cobas c 311 analyzer 3 Photometric principlesReaction cell and calibration data

Reaction cell and calibration data

The following three sections explain the Cell Blank Measurement report, the Working Information window, and information given on the Others tab. These three sections are frequently referred to in other parts of this document which describe result calculations of the various types of assays:

Both the Working Information window and the Others tab of the Utility > Application screen display calibration information for individual tests and calibrators, respectively. The Cell Blank Measurement report contains data necessary for the calculation of absorbance values, which are the basis for all other calculations.

e For more information, see:Cell Blank Measurement report on page B-21

Working Information window on page B-22

Others tab on page B-23

Cell Blank Measurement report

Reaction absorbance in a cell is measured against the cell's water blank value (current cell blank). This cell blank report is requested as part of weekly maintenance. The values on this report are stored and compared to the real time water blank values that display on the Reaction Monitor report.

e See Reaction monitor on page B-14.

If the difference between the current real time water blank values and the previous cell blank measured by the Cell Blank maintenance function is greater than 0.1 Abs, an alarm is issued.

Figure B-13 Example of a Cell Blank Measurement report

This report shows no abnormal cells.

Roche Diagnostics


3 Photometric principles cobas c 311 analyzerReaction cell and calibration data

Working Information window

h Calibration > Status > Calibration Result > Working Information

S1 Abs. The Working Information window displays the current calibration curve and values for the application selected under Calibration > Status > Calibration Result. For endpoint assays based on an RCM or Linear calibration, the value under S1 Abs. equals the blank calibrators absorbance value 104. For rate assays it is the rate of change in absorbance of the reaction with the blank calibrator. S1 Abs. is subtracted from the reaction absorbance of all other samples including calibrators Std(2) through Std(6), controls, STAT and routine samples.

K factor The K factoras well as S1 Abs.is used in the result calculation of every measured test. Given a linear calibration curve, the two main types of assays use the following formulas for result calculation:

Equation B-7 for endpoint assays

Equation B-8 for rate assays

On the Working Information window, K factors are always displayed as whole numbers. The correct decimal placement in a K factor depends on the decimal places in the concentration value for Std (1) displayed on the Others tab of the Utility > Application screen. If the Std (1) concentration has n decimal places, divide the displayed K factor by the n-th power of ten to obtain the correct value for result calculations.


Calibration factor

Absorbance after reaction is completed

Absorbance of Std (1)/blank calibrator (S1 Abs.)


Rate of change in absorbance of the reaction with the sample

Rate of change in absorbance of the reaction with Std (1)/blank calibrator

Cx K Ax Ab( ) Cb+=Cx K vx vb( ) Cb+=

K

Ax

Ab

Cb

vx

vb

Roche Diagnostics


cobas c 311 analyzer 3 Photometric principlesReaction cell and calibration data

Others tab

h Utility > Application > Others

Use this tab to display test parameters such as calibrator codes, calibrator set points, calibrator positions, and pipetting volumes.

When the tests Std (1) concentration (blank calibrator concentration) is programmed with a decimal, the displayed K factor on the Working Information window gets the same number of decimal places. This also determines the decimal placement in displayed results, as shown in the table below:

Figure B-15 Others tab on Utility > Application screen

Std (1) concentration

K (posted) K (calculations) Result

0 -1219 -1219 52

0.0 -12190 -1219.0 52.3

0.00 -121904 -1219.04 52.31

Table B-2 Determination of decimal placement

Roche Diagnostics


3 Photometric principles cobas c 311 analyzerEndpoint assays

Endpoint assays

In the following sections the various types of endpoint assays are explained in detail. After a brief listing of assay characteristics, a graphical representation of the absorbance in the course of the reaction is given, as well as an example of result calculation.

e For details on the various types of endpoint assays, see:1 Point assay on page B-24

2 Point End assay on page B-27

1 Point assay

Assay characteristics:

o Called 1 Point because only one measure point is designated in the Application screen.

o Addition of one or more reagents is possible.

o No sample blanking required.

o The absorbance reading for this type of assay can be taken during any disk rotation after addition of the final reagent.


Assay/Time/Point: [ 1 Point ] [ time ] [ ] [ 0 ] [ 0 ] [ 0 ]

c 311 1 571 time 10Cell blank = (C1 + C2 + C3) / 3

Reaction volume = 100-250 L

mp1

mp1

Roche Diagnostics


cobas c 311 analyzer 3 Photometric principlesEndpoint assays

1 Point assay graph

1 Point assay with R1 timing A graphic representation of a 1 Point assay using a reagent dispensed at R1 timing is shown below. The figure below shows an increase in absorbance as the reaction occurs. A decrease in absorbance as the reaction occurs is also possible.

1 Point assay with R1 andR2 or R3 timing

A graphic representation of a 1 Point assay using reagents dispensed at R1 and R2 or R3 timing is shown below.

Figure B-16 1 Point End assay - reagent at R1 timing

Abs

orba

nce

Time

S, R1

C1 C2 C3

mp1

Amp1

Figure B-17 1 Point End assay - reagents at R1 and R3 timing

, , ... The reaction cell's water blank values(a)


Pipetting of sample


, Pipetting of reagent at R2 or R3 timing

Measure point 1, endpoint (after reaction has reached equilibrium)

Absorbance at measure point 1

Abs

orba

nce

Time

S, R1

C1 C2 C3

Amp1

mp1

R2, R3

C1 C2

S

R1

R2 R3

mp1

Amp1

Roche Diagnostics



Sample program and calculations

This section gives an example of an applications result calculations.

e For more detailed explanations, see Comprehensive assay descriptions on page B-12.


The following data from Utility > Application are used for this calculation example:

Calculation of concentration The calculation of the concentration of the analyte in the sample uses the following equation:

Equation B-9

Applying these values to the above formulas (Equation B-9) yields:

Test CHO2I

Assay/Time/Point [ 1 Point ] [ 10 ] [ 57 ] [ 0 ] [ 0 ] [ 0 ]


Ser/Pl N000043 014 06/02/07 CELL 036 CHO2I 150.6ID 09:08:37

*** (PRIMARY)-(SECONDARY) ***CB1-3 1-10 11-20 21-30 31-40 41-50 51-57

922 3084 5112 5127 5120 5115 5110922 4771 5118 5127 5122 5114 5110924 5061 5121 5128 5120 5117 5108

5080 5122 5125 5119 5115 51085087 5124 5128 5119 5112 51085093 5124 5125 5118 5115 51065021 5126 5124 5120 5112 51065094 5128 5123 5116 51145104 5128 5123 5117 51105108 5128 5120 5117 5112


Symbol Definition Value

Absorbance value for concentration calculation (a)

(a) See Reaction Monitor report above.

0.5106

Concentration of the analyte in the sample

Calibration factor(b)

(b) Displayed on Working Information window. For explanations, see Working Information window on page B-22.

416.9

Absorbance of Std (1)/blank calibrator (S1 Abs.)(b) 0.1493

Concentration value for Std (1)/blank calibrator(c)

(c) Displayed on Utility > Application > Others. For explanations, see Others tab on page B-23.

0.0

, Instrument constants for a slope of 1 and an intercept of 0 1, 0

Table B-3 Definitions and values for quantities used in the calculation


Ax

Cx

K

Ab

Cb

IFA IFB

Cx 416,9 0,5106 0,1493( ) 0,0+ 416,9 0,3613= =Cx 150,6=

Roche Diagnostics



2 Point End assay

Assay Characteristics:

o Called 2 Point because there are readings at two measure points, and , which are designated on Utility > Application > Analyze.

o Allows for two or more reagent additions.

o Performs sample blank measurement.

o The first absorbance reading for this type of assay can be taken during any disk rotation. Usually it is taken before or shortly after the final reagent is added.

o The second absorbance reading can be taken during any disk rotation after the final reagent is added.


Assay/Time/Point: [ 2 Point End ] [ time ] [ ] [ ] [ 0 ] [ 0 ]

c 311 1 < 571 time 10Cell blank = (C1 + C2 + C3) / 3

Reaction volume = 100-250 L (at all measure points)

2 Point End assay graph

A graphic representation of a 2 Point End assay using reagents dispensed at R1 and R2 or R3 timing is shown below.

mp1 mp2

mp1 mp2

mp1 mp2

Figure B-19 2 Point End assay - reagents at R1 and R2 or R3 timing



Pipetting of sample

, Pipetting of reagent at R1 timing and of reagent at R2 or R3 timing

Measure point 1, sample blank (here before final reagent addition)

Measure point 2, endpoint (after reaction has reached equilibrium)


Abs

orba

nce

Time

S

C1 C2 C3

mp2

R2/R3

Amp2

mp1

R1

Amp1

C1 C2

S

R1 R2 R3mp1

mp2

Amp1 Amp2

Roche Diagnostics




This section provides an example of an applications result calculations.


The following data from the Utility > Application screen are used for this example:

The result calculation is based on a calculated value for the absorbance of the final reaction product . To determine this value the sample blank reading is corrected for dilution and subtracted:

Equation B-10 with

Assuming absorbance values on the reaction monitor report are the following:

Test GLUC2

Assay/Time/Point [ 2 Point End ] [ 10 ] [ 6 ] [ 24 ] [ 0 ] [ 0 ]

Ax

Ax Amp2 d Amp1=

dVsamp VR1+

Vsamp VR1 VR2+ +----------------------------------------------=


Ser/Pl N000001 001 11/01/07 CELL 055 GLUC2 5.3ID 13:53:33


1765 4608 4607 4605 4609 46141759 4611 4610 4610 4609 46121750 4607 4604 4610 4610 46082102 4603 4608 4608 4609 46073963 4610 4608 4612 46124474 4608 4607 4604 46094576 4609 4608 4607 4610



Absorbance value for concentration calculation

Absorbance at measure point 2 (24th measurement of cell)(a)


0.4607

Absorbance at measure point 1 (6th measurement of cell)(a) 0.1750

Dilution factor

Sample volume 2 L

Volume of reagent R1 150 L

Volume of reagent R2 50 L


Ax

Amp2

Amp1

d

Vsamp

VR1

VR2

Roche Diagnostics



The absorbance at measure point 1 is multiplied by the following to correct for dilution:

Therefore:

Calculation of concentration The calculation of the concentration of the analyte in the sample uses the following equation:

Equation B-11

Applying these values to the above formula (Equation B-11) yields:

(5.3 on reports and Data Review screen)

d 2L 150L+( )2L 150L 50L+ +( )---------------------------------------------------------

152202--------- 0,7525= = =

Ax 0,4607 0,7525 0,1750=Ax 0,4607 0,1317 0,3290= =



Calibration factor(a)

(a) Displayed on Working Information window. For explanations, see Working Information window on page B-22.

16.3

Absorbance value calculated above 0.3290

Absorbance of Std (1)/blank calibrator (S1 Abs.)(a) 0.0030

Concentration value for Std (1)/blank calibrator(b)

(b) Displayed on Utility > Application > Others. For explanations, see Others tab on page B-23.

0.0

, Instrument constants for a slope of 1 and an intercept of 0 1, 0



Cx

K

Ax

Ab

Cb

IFA IFB

Cx 16,3 0,3290 0,0030( ) 0,0+=Cx 16,3 0,3260( )=Cx 5,314=

Roche Diagnostics


3 Photometric principles cobas c 311 analyzerRate assays

Rate assays

The following sections explain in detail the various types of rate assays. After a brief listing of assay characteristics, a graphical representation of the absorbance in the course of the reaction is given, as well as an example of result calculation.

e For details on the various types of rate assays, see:Rate A assay on page B-30

Rate A assay with sample blank correction on page B-33

2 Point Rate assay on page B-36

Rate A assay


o One or more reagent additions are possible.

o Rate of change in absorbance is calculated by least squares method.

o Substrate depletion is monitored for linearity.


Assay/Time/Point: [ Rate A ] [ time ] [ ] [ ] [ 0 ] [ 0 ]

c 311 1 < 57 ; + 2 < ; 1 time 10Cell blank = (C1 + C2 + C3) / 3

Reaction volume = 100-250 L

Rate A assay graph

A graphic representation of a Rate A assay using a reagent dispensed at R1 is shown below.

Rate A assay with R1 timing

mp1 mp2

mp1 mp2 mp1 mp2

Figure B-21 Rate A assay - reagent at R1 timing

Abs

orba

nce

S, R1

C1 C2 C3

mp1 Timemp2

vx

Roche Diagnostics


cobas c 311 analyzer 3 Photometric principlesRate assays

Rate A assay with R1 andR2 or R3 timing

A graphic representation of a Rate A assay using reagents dispensed at R1 and R2 or R3 timing is shown below.


This section provides an example of an applications result calculations.



Figure B-22 Rate A assay - reagents at R1 and R2 or R3 timing



Pipetting of sample




Measure point 1 (initial measure point)

Measure point 2 (final measure point)

Abs

orba

nce

S, R1

C1 C2 C3

mp1 Timemp2

R2/R3

vx

C1 C2

S

R1

R2 R3

vx mp1 mp2

mp1

mp2

Test ALTL

Assay/Time/Point [ Rate A ] [ 10 ] [ 12 ] [ 31 ] [ 0 ] [ 0 ]

Roche Diagnostics



Calculation of concentration The calculation of the unknown concentration of the analyte in the sample uses the following equation:

Equation B-12 with




Ser/Pl N000001 063 16/02/07 CELL 58 ALTL 2.11ID 14:30:43

*** (PRIMARY)-(SECONDARY) ***CB1-3 1-10 11-20 21-30 31-40 41-50 51-573465 3505 19230 18231 17102 16511 159903467 3490 19130 18126 17008 16477 158853466 3489 19032 18026 16908 16410 15794

3486 18932 17933 16872 16377 15690 3478 18837 17822 16809 16303 15584 3477 18729 17610 16772 16278 1549519560 18638 17502 16703 16214 1539319503 18535 17396 16674 1617919420 18428 17306 16609 1611719328 18328 17201 16575 16084



Rate of change in absorbance of the reaction with the

sample

Rate of change in absorbance between (12th

measurement of cell) and (31st measurement)(a)



Calibration factor(b)

(b) Displayed on Working Information window. For explanations, see Working Information window on page B-22.

-42.04

Rate of change in absorbance of the reaction with the

sample

-0.0503/min

Rate of change in absorbance of the reaction with Std (1)/

blank calibrator(b)-0.0001/min

Concentration value for Std (1)/blank calibrator(c)

(c) Displayed on Utility > Application > Others. For explanations, see Others tab on page B-23.

0.00

, Instrument constants representing a slope of 1 and an

intercept of 0

1, 0


Cx K vx vb( ) Cb+[ ] IFA IFB+=vx v mp2 mp1( , )=

vx

v mp2 mp1( , ) mp1mp2

Cx

K

vx

vb

Cb

IFA IFB

Cx 42,04 0,0503 0,0001( )[ ] 0,0+{ } 1 0+=Cx 42,04 0,0502( )=Cx 2,110=

Roche Diagnostics



Rate A assay with sample blank correction


o Assay with sample blank measurement.

o One or more reagent additions are possible.

o Rate of change in absorbance is calculated by least squares method.

o Substrate depletion is monitored for linearity.


Assay/Time/Point: [ Rate A ] [ time ] [ ] [ ] [ ] [ ]

c 311 1 < < < 57( + 2) < ; ( + 2) < 1 time 10Cell blank = (C1 + C2 + C3) / 3Reaction volume = 100-250 L (at all measure points)

Rate A assay with sample blank graph

A graphic representation of a Rate A assay using a reagent dispensed at R1 and R3 timing is shown below.

mp1 mp2 mp3 mp4

mp3 mp4 mp1 mp2mp3 mp4 mp1 mp2

Figure B-24 Rate A assay with sample blank



Pipetting of sample

, Pipetting of reagent at R1 timing and of reagent at R3 timing

, Measure point 1 and 2 (initial and final measure points for rate reaction)

, Measure point 3 and 4 (initial and final measure points for sample blank)



Abs

orba

nce

S, R1

C1 C2 C3

mp1 Timemp2

R3

mp4mp3

v mp1 mp2( , )

v mp3 mp4( , )

C1 C2

S

R1 R3

mp1 mp2

mp3 mp4

v mp1 mp2( , ) mp1 mp2


Roche Diagnostics





Calculation of the rate of change in absorbance uses the following equation:

Equation B-13 with

The rate of change in absorbance between 15th and 23rd measurement of the reaction cell (sample blank) is multiplied by the following to correct for dilution:

Therefore:

Test CREJ2

Assay/Time/Points [ Rate A ] [ 10 ] [ 27 ] [ 37 ] [ 15 ] [ 23 ]

vx v mp2 mp1( , ) d v mp4 mp3( , )=

dVsamp VR1+

Vsamp VR1 VR2+ +----------------------------------------------=


Ser/Pl N000011 014 06/02/07 CELL 034 CREJ2 4.33ID 15:22:51

*** (PRIMARY)-(SECONDARY) ***CB1-3 1-10 11-20 21-30 31-40 41-50 51-60

356 955 847 816 1689 2002 2228356 915 844 812 1746 2017 2265356 883 841 809 1801 2048 2304

873 835 1092 1819 2063 2340867 832 1197 1854 2093 2372863 829 1365 1871 2107 2406860 826 1437 1905 2133 2440857 823 1504 1921 2147852 819 1568 1956 2178849 816 1632 1971 2189




Rate of change in absorbance between and 1.507 10-2 min-1

Rate of change in absorbance between and 3.093 10-2 min-1

Dilution factor

Sample volume 10 L

Reagent 1 volume 90 L

Reagent 2 volume 47 L


vx



d

Vsamp

VR1

VR2

d 10L 90L+10L 90L 47L+ +----------------------------------------------------

100147--------- 0,6803= = =

vx 0,0309 0,6803 0,0015( ) 0,0319= =

Roche Diagnostics




Equation B-14







136.16


(calculated above)

0.0319


blank calibrator(a)0.0001



0.00

, Instrument constants representing a slope of 1 and an

intercept of 0

1, 0



Cx

K

vx

vb

Cb

IFA IFB

Cx 136,16 0,0319 0,0001( )[ ] 0+=Cx 136,16 0,0318=Cx 4,3299=

Roche Diagnostics



2 Point Rate assay


o Rate assay measures rate of change in absorbance.

o Called 2 Point because there are 2 measure points (or duplicate readings at and ).

o The first absorbance reading for this type of assay can be taken during any disk rotation after the final reagent is added.

o This reaction is monitored for substrate depletion, but not for linearity.


Assay/Time/Point: [ 2 Point Rate ] [ time ] [ ] [ ] [ 0 ] [ 0 ]

c 311 1 < 571 time 10Cell Blank = (C1 + C2 + C3) / 3

Reaction volume = 100-250 L (at all measure points)

2 Point Rate assay graph - R1 and R2 or R3 timing

A graphic representation of a 2 Point Rate assay using reagents dispensed at R1 and R2 or R3 timing is shown below.

mp1mp2

mp1 mp2

mp1 mp2

Figure B-26 2 Point Rate assay - reagents at R1 and R2 or R3 timing



Pipetting of sample



, Measure point 1 and 2


Abs

orba

nce

S, R1

C1 C2 C3

mp1 Time

Amp2

mp2

Amp1R2/R3

C1 C2

S

R1

R2 R3

mp1 mp2

Amp1 Amp2

Roche Diagnostics





The result calculation is based on a calculated value for the rate of change in absorbance of the reaction mixture . To determine this value, readings are subtracted and divided by the time between measure points 1 and 2:

Equation B-15


Test CO2-L

Assay/Time/Point [ 2 Point Rate ] [ 10 ] [ 2 ] [ 18 ] [ 0 ] [ 0 ]


Ser/Pl N000002 064 16/02/07 CELL 039 CO2-L 24.2ID 15:52:23


7429 6721 6288 6007 5904 57947347 6665 6253 6000 5900 57797264 6619 6195 5988 5889 57687184 6569 6166 5977 5876 57577110 6522 6139 5967 58717036 6475 6113 5958 58606967 6434 6091 5946 5854

Figure B-27


Rate of change in absorbance

Absorbance at measure point 2 (a)

(a) See Reaction monitor above.

0.6522

Absorbance at measure point 1 0.7728

Time between and 3.440 min


vx

vx Amp2 Amp1( ) t=

vx

Amp2

Amp1

t mp1 mp2

vx 0,6522 0,7728( ) 3,440 0,0351= =

Roche Diagnostics




Equation B-16

Therefore:






-730.0

Rate of change in absorbance of the reaction with the sample -0.0351


blank calibrator(a)-0.0019



0.0

, Instrument constants for a slope of 1 and intercept of 0 1, 0



Cx

K

vx

vb

Cb

IFA IFB

Cx 730,0 0,0351 0,0019( )[ ] 0,0+=Cx 730,0 0,0332=Cx 24,24=

Roche Diagnostics


cobas c 311 analyzer 3 Photometric principlesProzone check

Prozone check

There are two prozone check methods available:

o Antigen readdition method

o Reaction rate method

Both of these methods can be applied to any type of assay.

e For more information, see:Antigen readdition method on page B-39

Reaction rate method on page B-42

Antigen readdition method

Prozone checks applying the antigen readdition method compare the absorbance before and after a final reagent addition at R2 or R3 timing, as indicated below:

Figure B-28 Prozone check - antigen readdition method



Pipetting of sample

, , Pipetting of reagent at R1, R2, and R3 timing

, Prozone measure points 1 and 2

, Absorbance at and

Abs

orba

nce

Time

S, R1

C1 C2 C3

pmp2

R3Apmp2

pmp1

Apmp1

R2

C1 C2

S

R1 R2 R3

pmp1 pmp2

Apmp1 Apmp2 pmp1 pmp2

Roche Diagnostics


3 Photometric principles cobas c 311 analyzerProzone check

Programming and calculation

Program a prozone check on the Analyze tab of the Utility > Application screen according to the following description:

To the right of the Prozone Limit field there are nine boxes:

[ lower limit ] [ upper limit ] [ ] [ ] [ 0 ] [ 0 ] [ comp. ] [ 0 ] [ 0 ]

o The first two boxes indicate the lower and upper prozone limits (in Abs 104).

o The next four boxes are for the prozone measure points ( ):

O 3rd entry: First prozone measure points ( )

O 4th entry: Second prozone measure points ( )

O 5th entry: Set to zero for this method

O 6th entry: Set to zero for this method

Appropriate values are: 2 < 57. If all entries are set to zero, prozone check is not performed.

o The seventh box (Inside/Outside) indicates in which case a data alarm (>Proz) is issued: If the entry is set to Inside, an alarm is issued in case the obtained check value lies inside the defined range between the lower and upper prozone limits (first two boxes). Vice versa, if the entry is set to Outside, an alarm is issued in case the obtained check value lies outside the defined range.

o The eighth and ninth boxes are not used (set to zero) for this method.

Figure B-29 Application parameters of an application with prozone check

pmp1 pmp2

pmp

pmp1pmp2

pmp1 pmp2

Roche Diagnostics



Prozone check value calculation The calculation of the prozone check value uses the following equation:

Equation B-17 with

Calculation example

This section provides an example of a 2 Point End assay with prozone check (antigen readdition method) with the calculation of the prozone check value.



Equation B-18 with

Prozone check value

Absorbance at prozone measure point 2

Absorbance at prozone measure point 1

Dilution factor

Sample volume

R1 volume

R2 volume

PC Apmp2 d Apmp1=

dVsamp VR1+

Vsamp VR1 VR2+ +----------------------------------------------=

PC

Apmp2

Apmp1

d

Vsamp

VR1

VR2

Test ALBU2

Assay/Time/Point [ 2 Point End ] [ 10 ] [ 6 ] [ 15 ] [ 0 ] [ 0 ]

Prozone Limit [ -32000 ] [ 1000 ] [ 24 ] [ 30 ] [ 0 ] [ 0 ] [ Inside ] [ 0 ] [ 0 ]


Prozone check value

Absorbance at prozone measure point 2 0.9951

Absorbance at prozone measure point 1 1.1070

Dilution factor (correcting for R3 addition)

Sample volume 6.0 L

R1 volume 100 L

R2 volume 20 L

R3 volume 26 L


PC Apmp2 d Apmp1=

dR3Vsamp VR1 VR2+ +

Vsamp VR1 VR2 VR3+ + +-------------------------------------------------------------=

PC

Apmp2

Apmp1

dR3

Vsamp

VR1

VR2

VR3

Roche Diagnostics


3 Photometric principles cobas c 311 analyzerProzone check

Applying these values to the above formulas (Equation B-18) yields:

Therefore:

The calculated prozone check value is compared to the lower and upper prozone limits on Utility > Application > Analyze. In the above calculated example the prozone check value is 0.0775 104 or 775. This value lies inside the defined prozone limits, and the seventh box is also set to Inside. Thus, a data alarm (>Proz) is issued: The test result is flagged on the Reaction Monitor, on the Data Review screen, and the prozone data alarm is printed on the patient report.

Reaction rate method

Prozone checks applying the reaction rate method compare the rate of change in absorbance at two different times after final reagent addition, as indicated below:

dR36,0L 100L 20L+ +( )

6,0L 100L 20L 26L+ + +( )---------------------------------------------------------------------------------126152--------- 0,8289= = =

PC Apmp2 d Apmp1=PC 0,9951 0,8289 1,1070 0,0775= =

Figure B-30 Prozone check - reaction rate method



Pipetting of sample



Prozone measure point n, with n = 1, 2, 3, and 4

Rate of change in absorbance between and

Absorbance difference between and

Abs

orba

nce

Time

S, R1

C1 C2 C3

R2/R3

pmp1

A pmp4 pmp3( , )

pmp2 pmp3 pmp4

v pmp1 pmp2( , )

v pmp3 pmp4( , )

A pmp2 pmp1( , )

C1 C2

S

R1

R2 R3

pmpn

v pmpn pmpm( , ) pmpn pmpm

A pmpn pmpm( , ) pmpn pmpm

Roche Diagnostics



Programming and calculation

To the right of the Prozone Limit field there are nine boxes:

[ lower limit ] [ upper limit ] [ ] [ ] [ ] [ ] [ comp. ] [ 0 ] [ 0 ]

o The first two boxes indicate the lower and upper prozone limits (in Abs 104).

o The next four boxes are for the prozone measure points ( ):

O 3rd entry: First prozone measure point ( )

O 4th entry: Second prozone measure point ( )

O 5th entry: Third prozone measure point ( )

O 6th entry: Fourth prozone measure point ( )

Appropriate values are: 1 < 57 and 1 < 57. If all entries are set to zero, prozone check is not performed.

o The seventh box (Inside/Outside) indicates in which case a data alarm (>Kin) is issued: If the entry is set to Inside, an alarm is issued in case the obtained check value lies inside the defined range between the lower and upper prozone limits (first two boxes). Vice versa if the entry is set to Outside, an alarm is issued in case the obtained check value lies outside the defined range.

o The eighth and ninth boxes define additional conditions for the reaction rate method. These allow you to neglect the prozone check in case the reaction rates get too low.

The entry in the eighth box defines the limit (in Abs 104) for the difference in absorbance between and . If the measured difference between these points falls below the limit, the prozone check is neglected.In other words:

If , reaction rate prozone check is not performed, where is defined in the eighth box

Likewise, the ninth box defines the limit between and . If the measured difference falls below the limit, the prozone check is neglected.In other words:

If , reaction rate prozone check is not performed, where is defined in the last box of the Prozone Limit line.


Equation B-19 with

pmp1 pmp2 pmp3 pmp4

pmp

pmp1pmp2

pmp3pmp4

pmp1 pmp2 pmp3 pmp4

pmp1 pmp2

Apmp2 Apmp1 F410 Application screen. If a measured serum index value is greater than the corresponding value in the L, H, or I box, an alarm is issued. When serum index limits are set to 0, the serum index check will be neglected.

The following example shows how a data alarm is issued when a test-specific limit value for a serum index is exceeded in a patient sample.

Example For this example the application GLUC2 is programmed with a L index limit of 10, H index limit of 10, and I index limit of 60.

Serum index Conventional units SI units

Lipemia index L 0-1000 mg/dL 0-11 mmol/L Intralipid

Hemolysis index H 0-1000 mg/dL 0-620 mol/L Hemoglobin

Icterus index I 0-60 mg/dL 0-1000 mol/L Total bilirubin

Table B-14

Note that these are just example values! Real values are downloaded or can be retrieved from package inserts.

Roche Diagnostics


4 Serum index principles cobas c 311 analyzerSerum index data alarms

If the obtained L or H index is greater than 10 and/or the I index is greater than 60, a data alarm (>Index) is issued.

The measurement of the sample yielded an L index of 31, H index of 0, and I index of 7. The results are displayed on the Data Review screen.

For GLUC2 the limit of 10 for the L index is exceeded. Therefore a >Index data alarm is attached to the result.

Figure B-32 Utility > Application > Range with limits set for all three serum indices

Figure B-33

5 ISE unit - Ion selective electrode calibration . . . . . . . . . . . . . . . C-3

6 Photometric calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-9

Calibration C

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COBI CD Version 1.0 C-3

cobas c 311 analyzer 5 ISE unit - Ion selective electrode calibrationTable of contents

ISE unit - Ion selective electrode calibration

This chapter provides you with an overview of the calibration of ion-selective electrode tests used by the cobas c 311 analyzer.

ISE calibration ............................................................................................................ C-5

Slope calculation ........................................................................................................ C-6

Internal standard calculation ..................................................................................... C-6

One-point calibration ................................................................................................ C-7

Compensation overview ............................................................................................ C-7

Compensation value calculation ............................................................................... C-7

Reference electrode ..................................................................................................... C-8


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C-4 COBI CD Version 1.0

5 ISE unit - Ion selective electrode calibration cobas c 311 analyzerTable of contents

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cobas c 311 analyzer 5 ISE unit - Ion selective electrode calibrationISE calibration

ISE calibration

The following ISE calibrators are used depending on the calibration method:

o Std (1) or S1: ISE Low, a water-based solution

o Std (2) or S2: ISE High, a water-based solution

o Std (3) or S3:

o For global use: ISE Comp., a serum-based solution, is used for full calibrations.

o For use in US only: ISE High (compensated) with compensated set points is used for full calibrations.

The following table displays all calibration methods and the corresponding calibrators.

Additionally, an internal standard (IS) will be measured. The calibration interval for all ISE applications is 24 hours.

For each calibration, the standard solutions are aspirated into the electrode cartridges, andafter equilibration occurs at the electrode membranethe electromotive force (EMF, voltage) is measured.

The slope of the calibration will be calculated based upon these readings and the assigned value of the standards.

Method Used ISE calibrators

Blank Only ISE Comp. (not recommended in US)

2 Point ISE Low and ISE High

Full For global use: ISE Low, ISE High, and ISE Comp.

For use in US only: ISE Low, ISE High, and ISE High (compensated)

Table C-1 Used ISE calibrators depending on the calibration method

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5 ISE unit - Ion selective electrode calibration cobas c 311 analyzerSlope calculation

Slope calculation

The slope is calculated in millivolts (mV) from the aqueous high and low standards. The slope is calculated according to the following formula:

Equation C-1

Due to factors such as the condition of the electrodes, the measured slope may deviate from this ideal slope. Therefore, the slope obtained should fall within the following ranges:

Internal standard calculation

In any ISE measurement system a number of junctions between wires, membranes, and reagents exist. The internal standard compensates for system-related variations.

After the slope is established during a calibration, the internal standard concentration is calculated. The concentration of Na+, K+, and Cl- in the internal standard is calculated from the electromotive force (EMF, voltage) of each electrode measured during calibration according to the formula below.

Equation C-2

The calculated value of the internal standard, as well as the voltage, is shown on the Calibration report.

Slope

EMF (voltage) of high standard

EMF (voltage) of low standard

Concentration of high standard

Concentration of low standard

Na+ 50 to 68 mV

K+ 50 to 68 mV

Cl- -40 to -68 mV

SEH EL

CHCL------ log

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

S

EH

EL

CH

CL

Concentration of the specific ion in the internal standard

Input concentration of the low standard

EMF (voltage) of the internal standard for the specific ion

EMF (voltage) of the low standard for the specific ion

Slope

CIS CL 10EIS EL( ) S=

CIS

CL

EIS

EL

S

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cobas c 311 analyzer 5 ISE unit - Ion selective electrode calibrationOne-point calibration

One-point calibration

An internal standard, labeled as ISE Internal Standard (IS), is measured during calibration as well as before and after each routine sample. These measurements are used to correct for system-related drifts (junction potential differences, differences in electrode conditions, and the like).

Compensation overview

Since the standard low and high are aqueous, a protein-based ISE Standard 3 is used for compensating the differences in the electrode response between aqueous solutions and a human serum matrix.

In the US, the ISE Standard High (compensated) with compensated set points is used for ISE Standard 3 to correct differences between aqueous standards and human serum matrix.

These differences are very small for regularly maintained ISE units. However, under certain conditions these differences may become more prominent, thus requiring compensation.

Compensation value calculation

The concentration of ions in the compensator is calculated according to the following formula:

Equation C-3

The formula for finding the compensation value (C. Value):

C. Value = assigned value (S3) - calculated value (S3)

This compensation value is automatically updated after each successful calibration.

During calibration the compensation value is compared to the compensation value of the previous calibration. If the percent difference is greater than the Compensated Limit on Utility > Application > Calib., a Cal.E alarm is issued.

The ISE unit is in an optimal condition when the compensation values (C. Value) are stable and negligible low.

Concentration of ions in the ISE Standard 3 (S3)

Concentration of the internal standard, determined during calibration

EMF (voltage) of the compensator for the specific ion

EMF (voltage) of the internal standard for the specific ion

Slope

S3Conc CISEC EIS( ) S10=

S3Conc

CIS

EC

EIS

S

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5 ISE unit - Ion selective electrode calibration cobas c 311 analyzerReference electrode

Reference electrode

A 1M KCl solution is measured concurrently with each sample analysis. The reference electrode is used for this purpose. The voltage of the reference electrode serves as a reference point for all measurements. That is, all reported voltages are readings from which the voltage of the reference electrode has been subtracted.

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cobas c 311 analyzer 6 Photometric calibrationTable of contents

Photometric calibration

This chapter provides you with an overview of the calibration types used by the cobas c 311 analyzer for photometric assays. The K factor, calibration updates and calculating results are also discussed.

Calibration checks .................................................................................................... C-11

Calibration overview ................................................................................................ C-14

Calibration types ................................................................................................ C-15

K factor ............................................................................................................... C-15

Calibration methods .......................................................................................... C-16

Blank calibration .......................................................................................... C-17

Span calibration ............................................................................................ C-17

2 Point calibration ........................................................................................ C-18

Full calibration .............................................................................................. C-18

Calibration update types .................................................................................... C-19

K factor calculation ............................................................................................ C-19

Introduction to weighting .................................................................................. C-21

Calculation without weighting .................................................................... C-21

Calculation with weighting .......................................................................... C-21

Weighting factors .......................................................................................... C-21

Linear calibration ..................................................................................................... C-22

Linear two-point calibration graph ................................................................... C-22

Linear two-point calculation ............................................................................. C-23

Assay types .......................................................................................................... C-24

RCM calibration ....................................................................................................... C-25

RCM calibration graph ...................................................................................... C-25

RCM calculation ................................................................................................. C-26

Assay types .......................................................................................................... C-26

RCM2T1 calibration ................................................................................................ C-27

RCM2T1 calibration graph ................................................................................ C-27

RCM2T1 calculation .......................................................................................... C-28

Assay types .......................................................................................................... C-28


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6 Photometric calibration cobas c 311 analyzerTable of contents

RCM2T2 calibration ................................................................................................ C-29

RCM2T2 calibration graph ................................................................................ C-29

RCM2T2 calculation .......................................................................................... C-30

Assay types .......................................................................................................... C-30

Spline calibration ..................................................................................................... C-31

Spline calibration graph ..................................................................................... C-31

Spline calculation ............................................................................................... C-32

Assay types .......................................................................................................... C-32

Line Graph calibration ............................................................................................. C-33

Line Graph calibration graph ............................................................................ C-33

Line Graph calculation .............................

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