Cobas-C-311-Operator Manual-En PDF
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cobas c 311 a an nalyzer COBI CD Compendium of Background Information
cobas cob as c 31 311 1 anal analyze yzer r
Document information Revision history
CO COBI BI CD
Edition for
Revision date
cobas c 311
April 2007
Changes
version
1.0
Edition notice
analyzer
cobas c
311 analyzer Compendium of Background Information
This document is for users of the t he 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 o ut 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
Copyright Trademarks
311 analyzer.
© 2007, Roche Diagnostics GmbH. All rights reserved. The following trademarks are acknowledged: COBAS, COBAS C, and LIFE NEEDS ANSWERS are trademarks t rademarks 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: 61010-1: 200 20011
o
IEC 61010-261010-2-010: 010: 2003
o
IEC 61010-261010-2-081: 081: 2001
o
IEC 61010-261010-2-101: 101: 2002
o
UL 61010-1: 61010-1: 2001 2001
o
CAN/CS CAN /CSA A C22.2 C22.2 No. No. 610 61010 10-1-1-04 04
o
EN 61326-261326-2-6:20 6:2006 06
Compliance is demonstrated by the following marks: 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. 105105-8717 8717 JAPAN JAPAN
Roche Diagnostics GmbH Sandhofer Strasse 116 D-68305 Mannheim Germany
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cobas cob as c 31 311 1 anal analyze yzer r
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cobas cob as c 31 311 1 analy analyze zer r
Table T able of contents Document information Contact addresses Table of contents How to use the CD
2 3 5 7
Installation of Adobe Acrobat Reader Where to find information Online Help system
7 7 8
Measurement technology 1
6
Part A
General photometer characteristics
ISE un unit it - Ion sele selectiv ctive e el electr ectrode ode princ principle iples s
Phot Photom omet etri ric c pr prin inci cipl ples es
Types of photometric assays Comprehensivee assay descriptions Comprehensiv Reaction cell and calibration data Endpoint assays Rate assays Prozone check Summary of assay techniques 4
5
B-9 B-12 B-21 B-24 B-30 B-39 B-44
Seru Serum m iind ndex ex prin princi cipl ples es
Introduction Definition of serum indices Measurement Measurem ent of serum indices Evaluating serum indices
B-49 B-49 B-49 B-51
Serum index data alarms
B-51
Calibration
Part C
C-25 C-27 C-29 C-31 C-33
Part D
Calc Calcul ulat atin ing g da data ta a ala larm rms s
Quality control 8
C-5 C-6 C-6 C-7 C-7 C-7 C-8
D-5 D-5 D-7 D-9 D-9 D-11 D-12 D-12
Part E
App pply lyin ing gQ QC C rul rules es
Introduction Rule 1: 1-2SD Rule 2: 1-2.5SD (Q2.5SD alarm) Rule 3: 1-3SD (Q3SD alarm) Rule 4: 2-2SA (S2-2Sa alarm) Rule 5: R-4SD (R4SD alarm) Rule 6: 2-2SW (S2-2Sw alarm) Rule 7: 4-1SA (S4-1Sa alarm)
E-5 E-6 E-6 E-7 E-8 E-9 E-10 E-11
Rule 8: 4-1SW (S4-1Sw alarm) Rule 9: 10XA (S10Xa alarm) Rule 10: 10XW (S10Xw alarm)
E-12 E-13 E-14
Index
ISE uni unitt - Ion Ion se select lective ive elec electro trode de calibr calibratio ation n
ISE calibration Slope calculation Internal standard calculation One-point calibration Compensation overview Compensation value calculation Referencee electrode Referenc
RCM calibration RCM2T1 calibration RCM2T2 calibration Spline calibration Line Graph calibration
Introduction Prozone effect Linearity verification (>Lin) Sensitivity limit check (Sens.E) Duplicate limit check (Dup.E) Technical limit check (>Test) (>Test) Repeat limit check (>Rept) Reaction limit check (>React)
Part B
Introduction B-5 Calculation of unknown sample concentrations B-5 3
C-11 C-14 C-22
A-5 7
2
Calibration checks Calibration overview Linear calibration
Calculating data alarms
Phot Photom omet etri ric c te tech chno nolo logy gy
T Test est principles
Phot Photom omet etri ric c cal calib ibra rati tion on
Index
Part F F-3
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cobas cob as c 31 311 1 analy analyze zer r
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 PDFformat and requires Adobe Acrobat Reader to be installed. If you do not have this software installed, refer to the instructions for f or the Installation of Adobe Acrobat Reader below. You You may access information by selecting a topic from f rom 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 i nstalled 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 AdbeRdr707_en_US.exe to start the installat installation ion 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 information The following documents are provided to assist in finding f inding desired information quickly: Operator’s Manual
Online Help
COBII CD COB
Contains information about safety, safet y, hardware 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. Contains a detailed description of the software of the cobas c 311 analyzer. In addition to the software description, the t he whole Operator’s Man Manual ual is included in the Online Help. This makes it possible to retrieve information from both Online Help and Operator’s Manual using the search functions available for electronically stored documents. The COBI CD (Compendium of Background Information) provides you you with background information about the technologies, test principles, their theory and calibration methods used by the cobas c 311 analyzer analyzer.. It also provides a complete glossary. The information can be read and printed using Adobe Acrobat Reader. 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 convenient way to find f ind information, such as explanations of screens and dialog boxes and how to perform particular parti cular 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.
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Measurement Measur ement technology
1
A
Phot Photom ometri etricc tec techn hnol ology ogy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-3 A-3
cobas c 311 analyzer
1 Photometric technology Table of contents
Photometric technology
This chapter provides you with an overview of the application applicati on of photometric technology in the cobas c 311 analyzer.
In this chapter
Chapter
1
General photometer characteristics ........................................................... ............................. ............................................. ............... A-5
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Table of contents
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1 Photometric technology General photometer characteristics
General photometer characteristics An illustration of the light path is shown below. A
B
C
D
E
F
G
H
I
J
K
M
L
N
A
Grating
F
Reaction cell and contents
K Infrared cut filter
B
Photometer
G
Incubator bath
L
C
Slit
H
Slit (in)
M Photometer lamp
D
Imaging lens
I
Condenser lens
N
E
Slit (out)
J
Mask
Fig Figure ure A-1
Water jacket Detector
Photometer lightpath
When the light beam enters the photometer, photometer, it strikes a diffraction grating, which separates the light into its constituent wavelength wavelengthss and reflects them onto a fixed fi xed array of 12 photodiodes. Each photodiode is permanently positioned positi oned 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, However, using the difference between readings at two wavelengths waveleng ths (bichromatic system) eliminates the effect of interferences sometimes found when using a single si ngle wavelength (monochromatic (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 absorb ance readings are taken 57 times (57 measure points) in 10 minutes. Choice of wavelengths
Bichromatic analysis uses two wavelengths: wavelengths: One that is at or near the peak absorbance of the chromogen produced by the reaction, and a second wavelength at which lit little tle or no absorbance of the desired chromogen occurs.
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General photometer characteristics
An Anyy absorbanc absorbancee ( A 2 ) that occur occurs, s, due to interfer interference ence from other substances substances in the sample, is measured at the secondary wavelength. This amount is then subtracted subtr acted from the total total ab absorban sorbance ce ( A1 ) occ occurring urring at the the primary primary wavele wavelength ngth to yield yield the net C absorbance ( A ).
A1
Observed
Chromophore
A
C
e c n a b r o s b A
A2 Interferent
λ1
λ2
Wavelength Fig Figure ure A-2
Bichromatic 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 ty pe programmed from the application parameters determine how final results are calculated for each test.
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Test T est principles
B
2
ISE unit - Ion selective electrode principles . . . . . . . . . . . . . . . . B-3
3
Phot Photom ometri etricc prin princi cipl ples es . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-7 B-7
4
Serum Serum index index princi principl ples es . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-47 B-47
cobas c 311 analyzer
2 IIS SE unit - Ion selective electrode principles Table of contents
ISE unit - Ion selective electrode principles
This chapter provides you with an overview of the ion i on selective electrode test principles and result calculation used by the cobas c 311 analyzer.
In this chapter
Chapter
2
Introduction ............................................................ ............................ .............................................................. ................................................... ..................... B-5 Calculation of unknown sample concentrations .................................... ...................................................... .................. B-5
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2 IS ISE unit - Ion selective electrode principles
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Table of contents
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2 IIS SE unit - Ion selective electrode principles Introduction
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. rat io. The EMF values of each sample aretogether converted todata mmol/L by a calculation algorithm that uses the EMF data with fromvalues 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. 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: Equatio Equ ation n B-1
S ( E s E IS s – IS ) ⁄ C s = C .Value .Value + C IS × 10
C s
Concentration Concen tration of the specific ion in sample
C .Value .Value
Compensation value
C IS
Concentration Concen tration of the internal standard
E s
Electromotivee force (voltage) of the unknown sample for the specific ion Electromotiv
E IS
Electromotivee force (voltage) of the internal standard for the specific ion Electromotiv
S
Slope
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2 IS ISE unit - Ion selective electrode principles
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Calculation of unknown sample concentrations
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Table 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.
In this chapter
Chapter
3
Types of photometric assays .................................. . ................................................................ .................................................... ..................... B-9 Assay types and measure points ............................................................ ............................. ............................................. .............. B-9 Displaying assay assay type and measure points ......................................... ............ ............................................ ............... B-11 Comprehensivee assay descriptions .................................... Comprehensiv ..... ............................................................ ...................................... ......... B-12 Example of a 2 Point End assay assay ................................................................ ................................. ........................................ ......... B-12 Example of a Rate A assay ................................ ............................................................... .................................................. ................... B-17 Reaction cell and calibration d data ata ......................................................... ............................. .............................................. .................. B-21 Cell Blank Measurement 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 ..................................... ..... ............................................................. .................................................. ..................... Sample program and calculations ...................................................... ........................... .................................... ......... 2 Point End assay ............................ ........................................................... ........................................................... ..................................... ......... 2 Point End assay graph .................................. .................................................................. ............................................. ............. Sample program and calculations ...................................................... ........................... .................................... ......... Rate assays ................................. .................................................................. ................................................................ ............................................... ................ Rate A assay .............................................................. .............................. .............................................................. ........................................... ............. Rate A assay graph ................................................... ................... .............................................................. ..................................... ....... Sample program and calculations ...................................................... ........................... .................................... ......... Rate A assay with sample blank correction .............................................. ................. ...................................... ......... Rate A assay with sample blan blank k graph ........................................................ ............................... ......................... Sample program and calculations ...................................................... ........................... .................................... ......... 2 Point Rate assay ........................................................... ............................. ........................................................... .................................... ....... 2 Point Rate assay graph - R1 and R2 or R3 timing .................................... Sample program and calculations ...................................................... ........................... .................................... ......... Prozone check ................................................................ ............................... ................................................................ ........................................... ............
B-25 B-26 B-27 B-27 B-28 B-30 B-30 B-30 B-31 B-33 B-33 B-34 B-36 B-36 B-37 B-39
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Antigen readdition method .... ................................. ........................................................ .............................................. ................... Programming and calculation ..... ..................................... ................................................................ ................................ Calculation example ..................................................................................... ......................................................... ............................ Reaction rate method ..................................... .................................................................. .................................................... ....................... Programming and calculation ..... ..................................... ................................................................ ................................ Summary of assay techniques ............................................................... ................................. ................................................. ...................
B-39 B-40 B-41 B-42 B-43 B-44
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3 Photometric principles Types of photomet photometric ric assays
Types T ypes of photometric assays There are two fundamental types of photometric photometr ic assays on this instrument: o
Endpoint assays
o
Rate assays
Measurements Measurem ents are taken by the t he 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 t ypes are divided in endpoint assays and rate assays: Fun unda dame ment ntal al assa assay y ttyp ype e
Assa Assay y ttyp ype e
Char Charac acte teri rist stic ic
Endpoint assays
1 Point
Endpoint assay programmed for a single measure point
2 Poi Point nt End End
Endp Endpoi oint nt assa assayy wi with th ssam ampl plee bl blan ank k
Rate A
Rate assay applying least squares method on multiple measure points
2 Point Point R Rate ate
Rat Ratee ass assay ay pr progr ogramm ammed ed fo forr two m meas easure ure p poin oints ts
Rate assays
T Table able B-1
Assay types
e
For more information on endpoint assays, see: 1 Point assay on on page B-24 2 Point End assay on on page B-27
e
For more information on rate assays, see: Rate A assay on on page B-30 Rate A assay with sample blank correction on page B-33 2 Point Rate assay on on page B-36
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Types of photometric assays
Measure points
Independent of the programmed application parameters, the photometer measures the absorbance of a reaction mixture in fixed fi xed 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 ( mp 1 an d mp 2 ).
Fig Figure ure B-1
Photometer measure points
In this example, the application parameters define the 6th photometer measure point eter measure point ( mp 24 ) to be mp 2 . In ( mp 6 ) to be mp 1 and the 24th photom other words, mp 6 of the instrument is set to be mp 1 of the test calculation, and mp 24 of the instrument is set to be mp 2 of the test calculation.
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3 Photometric principles Types of photomet photometric ric assays
Displaying assay type and measure m easure points The Analyze tab on the Utility > Application screen displays the assay type and measure points among other application parameters for a selected test.
Fig Figure ure B-2
Analyze tab on Utility > Application scree screen n
a To To view the as assay say type and measure measure points points for a test 1
Select Utility Utility > Applicati Application. on.
2
Select the test you want to view from the test list on the t he left side of the t he 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/P Assay/Time/Point oint text boxes on U Utility tility > Application > Analyze are shown as follows: Assay/Time/Point: Assay/Time/P oint: [ Assay Type ] [ time ] [ mp 1 ] [ mp 2 ] [ mp 3 ] [ mp 4 ]
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Comprehensive assay descriptions
Comprehensive Comprehensiv e 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 t he application parameters and result calculations. e
For extended and Example of a 2programming Point End assay oncalculation on page B-12 examples, see: Example of a Rate A assay on 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 means there are readings at two mp 2 : measure points, mp 1 and o
mp 1 is the sample blank which is measured before or shortly after the final
reagent is added. o
mp 2 measures the absorbance of the final reaction product; it is set after addition
of final reagent and after the reaction is i s 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.
R 2 e c n a b r o s b A
R 1
Am p 2
S
Am p 1 C1 C2 C3
mp 1
mp 2
Time
Fig Figure ure B-3
2 Point End assay graph
C1, C2, ...
The reaction cell's water blank values (a)
S
Pipetting of sample
R 1
Pipetting of reagent at R1 timing
R 2
Pipetting of reagent at R2 timing
mp 1
1st photometric measure point (sample blank)
mp 2
2nd photometric measure point (endpoint)
Am p 1 , Am p 2
Absorbances at measure point 1 and measure point 2
(a) See Cell Blank Measurement report on on page page B-21 B-21..
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3 Photometric principles Comprehensive assay descriptions
Example data
The following data from the Utility > Application screen screen are used for this example: example: T Test est
GLUC2
Assay type
2 Point End
Time
10 min
Points
6, 24
2nd wavelength
700 nm
Primary wavelength
340 nm
Conc. value value for for Std (1)
0.0
Entries on Utility Utility > Application > Analyze
Fig Figure ure B-4
Entries on Utility > Application > Analyze
In the later sections, the entries for the Assay/Time/Point Assay/Time/Point text boxes on U Utility tility > Application > Analyze are shown as follows: Assay/Time/Point: Assay/Time/P oint: [ 2 Point End ] [ 10 ] [ 6 ] [ 24 ] [ 0 ] [ 0 ] This means: o o
Dilution factor
The assay type is 2 Point End. 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 f irst and second reagents is determined by the 24th photometer measurement of the respective reaction cell.
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 corr ection for for the dilution dilution is taken into into account. account. A dilution factor factor ( d ) is calculated calculated as follows and applied to the sample + R1 absorbance: Equatio Equ ation n B-2
V + V R 1 d = ------------samp ----------------------------------V samp + V R 1 + V R 2 152 2 µ L + 150 µ L d = ---------------------------------------------------- = -------- = 0,7525 202 2 µ L + 150 µ L + 50 µ L
d
Dilution factor
V samp
Sample volume
V R 1
R1 volume
V R 2
R2 volume
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Comprehensive assay descriptions
The two measure points for this assay’s 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 below..
Reaction monitor
Reaction Monitor window of a 2 Point End assay
Fig Figure ure B-5
You can move the focus from one measure point to the next using the scroll bar below b elow 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: Reaction Monitor
Ser/Pl
N000001
001
11/01/07
ID
CELL 055
06/02/07
GL G LUC2
17:54
5.3
13:53:33
***
(PRIMARY) - (SECONDARY) ***
CB1-3
01-10
11-20
21-30
31-40
41-50
51-57
3239
1864
4601
4611
4606
4608
4611
3240
1819
4609
4610
4609
4609
4613
3240
1773
4607
4608
4611
4612
4610
1765
4608
4607
4605
4609
4614
1759
4611
4610
4610
4609
4612
1750
4607
4604
4610
4610
4608
2102
4603
4608
4608
4609
4607
3963
4610
4608
4612
4612
4474
4608
4607
4604
4609
4576
4609
4608
4607
4610
Fig Figure ure B-6
Reaction Monitor report
The values on the Reaction Monitor report (as well as those t hose in the Abs. field on the t he 4 Reaction Monitor window) are absorbance × 10 . Moreover, these values are already corrected for the water blank value, determined during the cell blank measurement. e
See Cell Blank Measurement report on on page B-21. 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.
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3 Photometric principles Comprehensive assay descriptions
Reaction absorbance
To dete determine rmine the reaction absorbance A x , the the sample blank value is corrected for dilution and then subtracted from the endpoint absorbance: Equatio Equ ation n B-3
A x = Am p 24 – d ⋅ A mp mp 6 A x = 0,4607 – 0,7525 ⋅ 0,1750 A x = 0,4 0,4607 – 0,1 ,13 317 = 0,3290
The absorb absorbanc ancee used used in in calc calcula ulatio tions ns ( A x ) is 0.3 0.329 290. 0. Calculation of concentration
The calculation of the unknown concentration of the analyte in the sample uses the following endpoint reaction formula: Equatio Equ ation n B-4
C x = [ K ( A x – Ab ) + C b ] ⋅ IF A + IF B
C x
Concentration Concen tration of the analyte (Gluc) in the sample
K
Calibration factor (also referred to as K factor)
A x
Absorbance after reaction is completed (calculated above: 0.3290)
A b
Absorbance of St Std d (1)/blank calibrator (S1 Abs.)
C b
Concentration Concen tration value for Std (1)/blank calibrator
IF A , IF B
Instrument constants representing representing a slope of 1 and an intercept of 0
K and A b are displayed on the Working Information window. Select Calibration >
Status > Calibratio Calibration n Result > Working Working Informatio Information n to display this window. window.
Fig Figure ure B-7
Working Information window
When the test's concentration 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. A b is the absorbance of the first standard standard solution, Std (1), which is a blank
calibrator. This value is also displayed di splayed on the Working Information window in the S1 Abs. field. e
See Working Information window on on page page B-2 B-222.
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cobas c 311 analyzer
Comprehensive assay descriptions
C b , the concentration of the analyte in the first standard solution Std Std (1), is displayed on the Others tab of the Utility > Application screen. This C b value controls the number of digits of the displayed K and the the rrounding ounding of the final results. results. W When hen the with a decimal, K includes an extra digit for each test's C b value is programmed
number to the right of the t he decimal point. e
Example values
See Others tab on page B-23. B-23.
The following values are used for this example: K
16.3 (displayed as 163 due to a Std (1) concentration value of 0.0)
A x
0.3290 (calculated above)
A b
0.0030 (displayed as 30 in the S1 Abs. field due to factor 104)
C b
0.0
IF A , IF B
Instrument constants representing representing a slope of 1 and an intercept of 0
Applying these values to the above formula C x = [ K ( A x – Ab ) + C b ] ⋅ IF A + IF B yields: C x = 16,3 ⋅ ( 0,3290 – 0,0030 ) + 0,0 C x = 16,3 ⋅ ( 0,3260 ) C x = 5,314
The result is rrounded ounded to 5.3 on the report because C b , the concentration value for Std (1), the blank calibrator, calibrator, contains one zero to the right of the decimal point as displayed displa yed on Uti Utility lity > Application Application > Others.
Roche Diagnostics B-16
COBI C D · Version 1.0
cobas c 311 analyzer
3 Photometric principles Comprehensive assay descriptions
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. t ime. 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, v : Equatio Equ ation n B-5
v x = -dA ------ x -dt
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 calculation—beginning with the reading at the first progra ogramm mmed ed meas easur uree p poi oin nt ( mp initial ) tth hrough ough th thee rrea eadi din ng at at tth he sec secon ond d programmed measure point ( mp final ). The absorbance absorbance values values are converte converted d into the rate of change change in absorbanc absorbancee ( v ) 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 ass ay using a reagent dispensed at R1 and R2 or R3 timing is shown below.
Absorbance limit
v x
e c n a b r o s b A
Blank S, R1
R2/R3
C1 C2 C3
mp 1
mp 2
Time
Fig Figure ure B-8
Rate A assay - reagents at R1 and R2 or R3 timing
C1, C2, ... S
The reaction cell's water blank values (a) Pipetting of sample
R1
Pipetting of reagent at R1 timing
R2/R3
Pipetting of reagent at R2/R3 timing
v x
mp 2 Rate of change in absorbance (slope) between mp 1 and
mp 1
First photometric measure point
mp 2
Last photometric measure point
(a) See Cell Blank Measurement report on on page page B-21 B-21..
Roche Diagnostics COBI C D · Version 1.0
B-17
3 Photometric principles
cobas c 311 analyzer
Comprehensive assay descriptions
Example data
The following data from Utility Utility > Application scree screen n are used for this example: example: T Test est
ALTL
Assay
Rate A
Time
10 min
Points
12, 31
2nd wavelength Primary wavelength
700 nm 340 nm
Conc. value value for for Std (1)
0.00
Entries on Utility Utility > Application > Analyze
Fig Figure ure B-9
Entries on Utility > Application > Analyze
In the later sections of this chapter, the entries for tthe he Assay/Time/P Assay/Time/Point oint text boxes on Utility Utili ty > Applic Application ation > Analyze ar aree shown as follows: follows: Assay/Time/Point: Assay/Time/P oint: [ Rate A ] [ 10 ] [ 12 ] [ 31 ] [ 0 ] [ 0 ] This means:
Reaction monitor
o o
The assay type is Rate A. 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.
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:
Fig Figure ure B-10 B-10
Reaction Monitor window of a Rate A assay
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 on page B-21. B-21.
Roche Diagnostics B-18
COBI C D · Version 1.0
cobas c 311 analyzer
3 Photometric principles Comprehensive assay descriptions
The absorbance values measured between the initial and the final absorbance reading ugh ( mp 12 th thro roug h mp 31 ) re repr pres esen entt a ch chan ange ge over over 4. 4.05 05 mi minu nute tes. s. The The math mathem emati atica call analysis results in a rate of change in absorbance of -0.0503 per minute. Reaction Monitor
Ser/Pl
N000001
063
16/02/07
ID
16/02/07
CELL 58
ALTL
16:22
2.11
14:30:43
***
(P PR RI M MA AR Y Y) )-( (SE C CO ON D DA AR RY Y)
CB1-3
1-10
** *
11-20
21-30
31-40
41-50
51-57
3465
3505
19230
18231
17102
16511
15990
3467
3490
19130
18126
17008
16477
15885
3466
3489
19032
18026
16908
16410
15794
3486
18932
17933
16872
16377
15690
3478
18837
17822
16809
16303
15584
3477
18729
17610
16772
16278
15495 15393
19560
18638
17502
16703
16214
19503
18535
17396
16674
16179
19420
18428
17306
16609
16117
19328
18328
17201
16575
16084
Reaction Monitor report
Figure Figu re B-11
Result calculation
The calculation of the unknown concentration of the analyte in the sample uses the following rate reaction formula: Equatio Equ ation n B-6
C x = [ K ( v x – v b ) + C b ] ⋅ IF A + IF B
K
Calibration factor
v x
Rate of change in absorbance (expressed in 104/min)
v b
Rate of change in absorbance of the reaction with Std (1)/blank calibrator
C b
Concentration Concen tration value for Std (1)/blank calibrator
C x
Concentration Concen tration of the analyte (AL (ALT) T) in tthe he sample
IF A , IF B
Instrument constants constants for a slope of 1 and an intercept of 0
K and v b are displayed on the Working Information window. Select Calibration >
Status > Calibratio Calibration n Result > Working Working Informatio Information n to display this window. window.
Roche Diagnostics COBI C D · Version 1.0
B-19
3 Photometric principles
cobas c 311 analyzer
Comprehensive assay descriptions
Fig Figure ure B-12
Working Information window
When the test's concentration concentration value for Std (1) is programmed with a decimal, the displayed K factor includes extra digits for each number to the right of tthe he decimal point. v b is displayed in the S1 Abs. field of the Working Information window. e
See Working Information window on on page page B-2 B-222.
b , the concentration of the analyte in the C b the first standard solution, Std (1), is displayed on the Others tab of the Utility > Application scre screen. en. e
Example values
See Others tab on page B-23. B-23.
The following values are used for this example: K
-42.04 (displayed as -4204 due to a Std (1) concen concentration tration value of 0.00)
v x
-0.0503/min (calculated by least squares method)
v b
-0.0001/min (displayed (displayed as -1 in the S1 Abs. field due to factor 104)
C b
0.00
IF A , IF B
Instrument constants constants for a slope of 1 and an intercept of 0
Applying these values to the rate reaction formula (Equ ( Equatio ation n B-6) B-6) yields: C x = { –42,04 ⋅ [ – 0,0503 – ( – 0,0001 ) ] + 0,0 } ⋅ 1 + 0 C x = – 42,04 ⋅ ( –0,0502 ) C x = 2,110
The result is d displayed isplayed as 2.11 on the report because because C b , the concentration value for Std (1), the blank calibrator, calibrator, contains two zeroes to the right of the decimal point as displayed displa yed on Uti Utility lity > Application Application > Others. Others.
Roche Diagnostics B-20
COBI C D · Version 1.0
cobas c 311 analyzer
3 Photometric principles Reaction cell and calibration data
Reaction cell and calibration data The following three sections explain the Cell Blank Measurement 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 Working Information window and the Others tab of the Utility > Application screen display calibration information for individual tests and calibrators, respectively. respectively. The Cell Blank Measurement report contains data necessary for the calculation of absorbance values, which are the basis ba sis for all other calculations. e
For more information, see: Cell Blank Measurement report on on page B-21 Working Information window on on page B-22 Others tab on page page B-2 B-233
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 on page page B-1 B-144.
If the difference between the current real time water blank bl ank values and the previous cell blank measured by the Cell Blank maintenance maintenance function is greater than 0.1 Abs, an alarm is issued.
Fig Figure ure B-13
Example of a Cell Blank Measurement report
This report shows no abnormal cells.
Roche Diagnostics COBI C D · Version 1.0
B-21
3 Photometric principles
cobas c 311 analyzer
Reaction cell and calibration data
Working Workin g Information window h Calibr Calibration ation > Status > Calibrati Calibration on Result Result > Working Working Informa Information tion
Fig Figure ure B-14 B-14
S1 Abs.
Working Information window
The Working 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 calibrator’s calibr ator’s 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 t hrough Std(6), controls, STA STAT T and routine samples.
K factor
The K factor—as well as S1 Abs.—is used in the result calculation of every measured measured test. Given a linear calibration curve, the two main types of assays use the following formulas for result calculation: Equatio Equ ation n B-7
C x = K ⋅ ( A x – Ab ) + C b for endpoint assays
Equatio Equ ation n B-8
C x = K ⋅ ( v x – v b ) + C b for rate assays
K
Calibration factor
A x
Absorbance after reaction is completed
A b
Absorbance of Std Std (1)/blank calibrator (S1 A Abs.) bs.)
C b
Concentration Concen tration value for Std (1)/blank calibrator
v x
Rate of change in absorbance of the reaction with the sample
v b
Rate of change in absorbance of the reaction with Std (1)/blank calibrator
On the Working Working Information window, K factors are always displayed as whole numbers. The correct decimal placement placement in a K factor depends on the decimal places in the concentration concentration value for Std (1) displayed on the Others tab of the Utility 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 t he correct value for result calculations.
Roche Diagnostics B-22
COBI C D · Version 1.0
cobas c 311 analyzer
3 Photometric principles Reaction cell and calibration data
Others tab h Utili Utility ty > A Applica pplication tion > Others Others
Fig Figure ure B-15
Others tab on on Utility > Application scre screen en
Use this tab to display test parameters such as calibrator codes, calibrator set points, calibrator positions, and pipetting volumes. When the test’s test’s Std (1) concentration (blank calibrator concentration) 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: Std Std (1)
K (posted)
K (calculations)
Result
0
-1219
-1219
52
0.0
-12190
-1219.0
52.3
0.00
-121904
-1219.04
52.31
concentration
T Table able B-2
Determination of decimal placement
Roche Diagnostics COBI C D · Version 1.0
B-23
3 Photometric principles
cobas c 311 analyzer
Endpoint 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 on page B-24 2 Point End assay on 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 ty pe of assay can be taken during any disk rotation after addition of the final reagent.
Entries on Utility Utility > Application > Analyze c 311
Assay/Time/Point: Assay/Time/P oint: [ 1 Point ] [ time ] [ mp 1 ] [ 0 ] [ 0 ] [ 0 ] 1 ≤ mp 1 ≤ 57 1 ≤ time ≤ 10 Cell blank = (C1 + C2 + C3) / 3 Reaction volume = 100-250 µL
Roche Diagnostics B-24
COBI C D · Version 1.0
cobas c 311 analyzer
3 Photometric principles Endpoint 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. p ossible.
e c n a b r o s b A
S, R1
Am p 1
C1 C2 C3 Time
mp 1 Fig Figure ure B-16 B-16
1 Point assay with R1 and R2 or R3 timing
1 Point End assay - reagent at R1 timing
A graphic representation of a 1 Point assay using reagents dispensed at R1 and R2 or R3 timing is shown below.
e c n a b r o s b A
S, R1
R2, R3
Am p 1
C1 C2 C3
mp 1
Time
Figure Figur e B-17
1 Point End assay - reagents at R1 and R3 timing
C 1 , C 2 , ...
The reaction cell's water blank values (a)
S
Pipetting of sample
R 1
Pipetting of reagent at R1 timing
R 2 , R 3
Pipetting of reagent at R2 or R3 timing
mp 1
Measure point 1, endpoint (after reaction has reached equilibrium)
Am p 1
Absorbance at measure point 1
(a) See Cell Blank Measurement report on on page page B-21 B-21..
Roche Diagnostics COBI C D · Version 1.0
B-25
3 Photometric principles
cobas c 311 analyzer
Endpoint assays
Sample program and calculations
This section gives an example of an application’s result calculations. e
Entries on Utility Utility > Application > Analyze
For more detailed explanations, see Comprehensive assay descriptions on page B-12 B-12..
The following data from Utility Utility > Application are used for this calculation example: CHO2I [ 1 Point ] [ 10 ] [ 57 ] [ 0 ] [ 0 ] [ 0 ]
T Test est Assay/Time/Point
Reaction Monitor
Ser/Pl
N000043
014
06/02/07
ID
06/02/07
CELL 036
CHO2I
0 09 9:25
150.6
09:08:37
***
(P PR RI M MA AR Y Y) )-( (SE C CO ON D DA AR RY Y)
CB1-3
** *
1-10
11-20
21-30
31-40
41-50
51-57
922
3084
5112
5127
5120
5115
5110
922
4771
5118
5127
5122
5114
5110
924
5061
5121
5128
5120
5117
5108
5080
5122
5125
5119
5115
5108
5087
5124
5128
5119
5112
5108
5093
5124
5125
5118
5115
5106
5021
5126
5124
5120
5112
5106
5094
5128
5123
5116
5114
5104
5128
5123
5117
5110
5108
5128
5120
5117
5112
Fig Figure ure B-18 B-18
Reaction Monitor report
Calculation of concentration
The calculation of the concentration of the analyte in the sample uses the following fo llowing equation: Equatio Equ ation n B-9
C x = [ K ( A x – Ab ) + C b ] ⋅ IF A + IF B
Symbol
Definition
Value
A x
Absorbance value for conce concentration ntration calculation (a)
0.5106
C x
Concentration Concen tration of the analyte in the ssample ample
K
Calibration factor(b)
416.9
A b
Absorbance of Std Std (1)/blank calibrator (S1 A Abs.) bs.)(b)
0.1493
C b
Concentration Concen tration value for Std (1)/blank calibrator(c)
0.0
IF A , IF B
In Instr strum umen entt co cons nsta tant ntss fo forr a sl slop opee of 1 and and an iint nter erce cept pt o off 0
1, 0
T Table able B-3
Definitions and values for quantities used in the calculation
(a) See Re Reaction action Monitor report above. (b) Displayed on Working Working Inform Information ation window. window. For explanation explanations, s, see Working Information window on on page page B-2 B-222. (c) Displ Displayed ayed on Utility Utility > Applic Application ation > Others. Others. For expla explanati nations, ons, see Others tab on page page B-23 B-23..
Applying these values to the above formulas (Equ (Equatio ation n B-9) B-9) yields: C x = 416,9 ⋅ ( 0,5106 – 0,1493 ) + 0,0 = 41 416, 6,9 9 ⋅ 0, 0,36 3613 13 C = 150,6 x
Roche Diagnostics B-26
COBI C D · Version 1.0
cobas c 311 analyzer
3 Photometric principles Endpoint assays
2 Point End assay Assay Characteristics:
Entries on Utility Utility > Application > Analyze c 311
o
mp 2 , Called 2 Point because there are readings at two measure points, mp 1 and which are designa which designated ted on Utility > Application Application > Analyz Analyze. e.
o
Allows for two or more reagent additions.
o
Performs sample blank measurement.
o
The first absorbance reading for this type t ype 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: Assay/Time/P oint: [ 2 Point End ] [ time ] [ mp 1 ] [ mp 2 ] [ 0 ] [ 0 ] mp 2 ≤ 57 1 ≤ mp 1 < 1 ≤ time ≤ 10
Cell blank = (C1 + C2 + C3) / 3 Reaction volume = 100-250 µL (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.
R2/R3 e c n a b r o s b A
R1
Am p 2
S
Am p 1
C1 C2 C3
mp 1
mp 2
Time
Fig Figure ure B-19 B-19
2 Point End assay - reagents at R1 and R2 or R3 timing
C 1 , C 2 , ...
The reaction cell's water blank values (a)
S
Pipetting of sample
R 1 , R 2 ⁄ R 3
Pipetting of reagent at R1 timing and of reagent at R2 or R3 timing
mp 1
Measure point 1, sample blank (here before final reagent addition)
mp 2
Measure point 2, endpoint (after reaction has reached equilibrium)
Am p 1 , Am p 2
Absorbances at measure point 1 and measure point 2
(a) See Cell Blank Measurement report on on page page B-21 B-21..
Roche Diagnostics COBI C D · Version 1.0
B-27
3 Photometric principles
cobas c 311 analyzer
Endpoint assays
Sample program and calculations
This section provides an example of an application’s result calculations. e
For more detailed explanations, see Comprehensive assay descriptions on page B-12 B-12..
The following data from the Utility > Application screen screen are used for this example: example: T Test est
GLUC2
Assay/Time/Point
[ 2 Point End ] [ 10 ] [ 6 ] [ 24 ] [ 0 ] [ 0 ]
The result calculation is based on a calculated value for the absorbance of the ffinal inal reaction product A x . To To determine determine this value value the sample blank reading is corrected corrected for dilution and subtracted: Equatio Equ ation n B-1 B-10 0
A x = Am p 2 – d ⋅ A mp mp 1 with V + V R 1 d = ------------samp ----------------------------------V samp + V R 1 + V R 2
Reaction
Ser/Pl
N000001
001
11/01/07
ID
Monitor
CELL 055
06/02/07
GL G LUC2
17:54
5.3
13:53:33
***
(PRIMARY) - (SECONDARY) ***
CB1-3
01-10
11-20
21-30
31-40
41-50
51-57
3239
1864
4601
4611
4606
4608
4611
3240
1819
4609
4610
4609
4609
4613
3240
1773
4607
4608
4611
4612
4610
1765
4608
4607
4605
4609
4614
1759
4611
4610
4610
4609
4612
1750
4607
4604
4610
4610
4608
2102
4603
4608
4608
4609
4607
3963
4610
4608
4612
4612
4474
4608
4607
4604
4609
4576
4609
4608
4607
4610
Figure Figu re B-20
Reaction Monitor report
Assuming absorbance values on the reaction monitor report are the following: Symbol
Definition
A x
Absorbance value for concentration calculation
Am p 2
Absorbance at measure point 2 (24th measurement of cell) (a) 0.4607
Am p 1
Absorbance at measure point 1 (6th measurement of cell) (a)
d
Dilution factor
V samp
Sample volume
2 µL
V R 1
Volume of reagent R1
150 µL
V R 2
Volume of reagent R2
50 µL
T Table able B-4
Value
0.1750
Definitions and values for quantities used in the calculation
(a) See Re Reaction action Monitor report above.
Roche Diagnostics B-28
COBI C D · Version 1.0
cobas c 311 analyzer
3 Photometric principles Endpoint assays
The absorbance at measure point 1 is multiplied by the following to correct for dilution:
( 2 µ L + 150 µ L ) 152 d = --------------------------------------------------------- = -------- = 0,7525 ( 2 µ L + 150 µ L + 50 µ L ) 202 Therefore: A x = 0,4607 – 0,7525 ⋅ 0,1750 A x = 0,46 ,4607 – 0,1317 = 0,3290
Calculation of concentration
The calculation of the concentration of the analyte in the sample uses the following fo llowing equation: Equatio Equ ation n B-11
C x = [ K ( A x – Ab ) + C b ] ⋅ IF A + IF B
Symbol
Definition
C x
Concentration Concen tration of the analyte in the ssample ample
K
Calibration factor(a)
16.3
A x
Absorbance value calculated above
0.3290
A b
Absorbance of Std Std (1)/blank calibrator (S1 A Abs.) bs.)(a)
0.0030
C b
Concentration Concen tration value for Std (1)/blank calibrator(b)
0.0
IF A , IF B
In Instr strum umen entt co cons nsta tant ntss fo forr a sl slop opee of 1 and and an iint nter erce cept pt o off 0
1, 0
T Table able B-5
Value
Definitions and values for quantities used in the calculation
(a) Displayed on W Working orking Information Information window. window. For explana explanations, tions, see Working Information window on on pagee B-2 pag B-222. (b) Displayed on Utility > Application Application > Oth Others. ers. For explanation explanations, s, see Others tab on page page B-23 B-23..
Applying these values to the above formula (Equat ( Equation ion B-11) B-11) yields: C x = 16,3 ⋅ ( 0,3290 – 0,0030 ) + 0,0 C x = 16,3 ⋅ ( 0,3260 ) C x = 5,314 (5.3 on reports and Data Review screen)
Roche Diagnostics COBI C D · Version 1.0
B-29
3 Photometric principles
cobas c 311 analyzer
Rate 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 on page B-30 Rate A assay with sample blank correction on page B-33 2 Point Rate assay on on page B-36
Rate A assay Assay Characteristics:
Entries on Utility Utility >
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. linearit y.
Assay/Time/Point: Assay/Time/P oint: [ Rate A ] [ time ] [ mp 1 ] [ mp 2 ] [ 0 ] [ 0 ]
Application > Analyze c 311
1 ≤ mp 1 < mp 2 ≤ 57 ; mp 1 + 2 < mp 2 ; 1 ≤ time ≤ 10 Cell blank = (C1 + C2 + C3) / 3 Reaction volume = 100-250 µL
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
v x e c n a b r o s b A
S, R 1
C1 C2 C3
mp 1 Fig Figure ure B-21
mp 2
Time
Rate A assay - reagent at R1 timing
Roche Diagnostics B-30
COBI C D · Version 1.0
cobas c 311 analyzer
3 Photometric principles Rate assays
Rate A assay with R1 and R2 or R3 timing
A graphic representation of a Rate A assay using reagents dispensed at R1 and R2 or R3 timing is shown below.
v x
e c n a b r o s b A
S, R 1
R 2 /R 3
C1 C2 C3
mp 1
mp 2
Time
Fig Figure ure B-22
Rate A assay - reagents at R1 and R2 or R3 timing
C 1 , C 2 , ...
The reaction cell's water blank values (a)
S
Pipetting of sample
R 1
Pipetting of reagent at R1 timing
R 2 , R 3
Pipetting of reagent at R2 or R3 timing
v x
mp 2 Rate of change in absorbance (slope) between mp 1 and
mp 1
Measure point 1 (initial measure point)
mp 2
Measure point 2 (final measure point)
(a) See Cell Blank Measurement report on on page page B-21 B-21..
Sample program and calculations
This section provides an example of an application’s result calculations. e
For more detailed explanations, see Comprehensive assay descriptions on page B-12 B-12..
The following data from the Utility > Application screen screen are used for this example: example: T Test est
ALTL
Assay/Time/Point
[ Rate A ] [ 10 ] [ 12 ] [ 31 ] [ 0 ] [ 0 ]
Roche Diagnostics COBI C D · Version 1.0
B-31
3 Photometric principles
cobas c 311 analyzer
Rate assays
Reaction Monitor
Ser/Pl
N000001
063
16/02/07
ID
16/02/07
CELL 58
ALTL
16:22
2.11
14:30:43
***
(P PR RI M MA AR Y Y) )-( (SE C CO ON D DA AR RY Y)
CB1-3
1-10
** *
11-20
21-30
31-40
41-50
51-57
3465
3505
19230
18231
17102
16511
15990
3467
3490
19130
18126
17008
16477
15885
3466
3489
19032
18026
16908
16410
15794
3486
18932
17933
16872
16377
15690
3478
18837
17822
16809
16303
15584
3477
18729
17610
16772
16278
15495
19560
18638
17502
16703
16214
15393
19503
18535
17396
16674
16179
19420
18428
17306
16609
16117
19328
18328
17201
16575
16084
Figure Figu re B-23
Reaction Monitor report
Calculation of concentration
The calculation of the unknown concentration of the analyte in the sample uses the following equation: Equatio Equ ation n B-12
C x = [ K ( v x – v b ) + C b ] ⋅ IF A + IF B with v x = v (mp 2 mp , 1)
Symbol
Definition
v x
Rate of change in absorbance of the reaction with the sample
v (mp 2, mp 1)
Rate of change in absorbance between mp 1 (12th measurementt of cell) and mp 2 (31st measurement)(a) measuremen
C x
Concentration Concen tration of the analyte in the ssample ample
K
Calibration factor(b)
-42.04
v x
Rate of change in absorbance of the reaction with the sample
-0.0503/min
b v b
Rate of change in absorbance absorbance of the reaction with Std (1)/ blank calibrator(b)
-0.0001/min
C b
Concentration Concen tration value for Std (1)/blank calibrator(c)
0.00
IF A , IF B
Instrument constants representing representing a slope of 1 and an intercept of 0
1, 0
T Table able B-6
Value
Definitions and values for quantities used in the calculation
(a) See Re Reaction action Monitor report above. (b) Displayed on Working Working Inform Information ation window. window. For explanation explanations, s, see Working Information window on on page page B-2 B-222. (c) Displ Displayed ayed on Utility Utility > Applic Application ation > Others. Others. For expla explanati nations, ons, see Others tab on page page B-23 B-23..
Applying these values to the above formula (Equat ( Equation ion B-12) B-12) yields: C x = { –42,04 ⋅ [ – 0,0503 – ( – 0,0001 ) ] + 0,0 } ⋅ 1 + 0 C x = – 42,04 ⋅ ( –0,0502 ) C x = 2,110 (2.11 on reports and Data Dat a Review screen)
Roche Diagnostics B-32
COBI C D · Version 1.0
cobas c 311 analyzer
3 Photometric principles Rate assays
Rate A assay with sample blank correction Assay Characteristics:
Entries on Utility Utility > Application > Analyze c 311
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. linearit y.
Assay/Time/Point: Assay/Time/P oint: [ Rate A ] [ time ] [ mp 1 ] [ mp 2 ] [ mp 3 ] [ mp 4 ] 1 ≤ mp 3 Application screen screen are used for this example: example: T Test est
CO2-L
Assay/Time/Point
[ 2 Point Rate ] [ 10 ] [ 2 ] [ 18 ] [ 0 ] [ 0 ] Reaction Monitor
Ser/Pl
N000002
064
16/02/07
ID
16/02/07
CELL 039
CO2-L
16:14
24.2
15:52:23
** *** *
(P (PRI RIMA MARY RY) ) -
(S (SEC ECON ONDA DARY RY) )
** *** *
CB1-3
1-10
11-20
21-30
31-40
41-50
51-57
766
7824
6901
765
7728
6838
6396
6064
5934
5837
6355
6044
5925
5824
761
7522
6782
6318
6020
5915
5809
7429
6721
6288
6007
5904
5794
7347
6665
6253
6000
5900
5779
7264
6619
6195
5988
5889
5768
7184
6569
6166
5977
5876
5757
7110
6522
6139
5967
5871
7036
6475
6113
5958
5860
6967
6434
6091
5946
5854
Fig Figure ure B-27 B-27
The result calculation is based on a calculated value for the rate of change in absorbance of the reaction mixture mixture v x . T To o determine determine this value, readings are subtracted and divided by the time t ime between measure points 1 and 2: Equatio Equ ation n B-15
v x = ( Am p 2 – Am p 1 ) ⁄ t
Symbol
Definition
v x
Rate of change in absorbance
Am p 2
Absorbance at measure point 2 (a)
0.6522
Am p 1
Absorbance at measure point 1
0.7728
t T Table able B-9
Time between mp 1 and mp 2
Value
3.440 min
Definitions and values for quantities used in the calculation
(a) See Rea Reaction ction m monit onitor or abo above. ve.
Applying these values to the above formula (Equat ( Equation ion B-15) B-15) yields: v x = ( 0,652 6522 – 0,7728 ) ⁄ 3,440 = – 0,0351
Roche Diagnostics COBI C D · Version 1.0
B-37
3 Photometric principles
cobas c 311 analyzer
Rate assays
Calculation of concentration
The calculation of the unknown concentration of the analyte in the sample uses the following equation: Equatio Equ ation n B-1 B-16 6
C x = [ K ( v x – v b ) + C b ] ⋅ IF A + IF B
Symbol
Definition
C x
Concentration Concen tration of the analyte in the ssample ample
K
Calibration factor(a)
v x
Rate of of change change in abso absorbanc rbancee of the re reactio action n with th thee sample -0.0 -0.0351 351
v b
Rate of change in absorbance absorbance of the reaction with Std (1)/ (a) blank calibrator
-0.0019
C b
Concentration Concen tration value for Std (1)/blank calibrator(b)
0.0
IF A , IF B
In Inst stru rume ment nt cons consta tant ntss for for a slo slope pe of 1 aand nd in inte terc rcep eptt of of 0
1, 0
T Table able B-10
Definitions and values for quantities used in the calculation
Value
-730.0
(a) Displayed on W Working orking Information Information window. window. For explana explanations, tions, see Working Information window on on page page B-2 B-222. (b) Displayed on Utility > Application Application > Oth Others. ers. For explanation explanations, s, see Others tab on page page B-23 B-23..
Therefore: C x = – 730,0 ⋅ [ – 0,0351 – ( –0,0019 ) ] + 0,0 C x = – 730,0 ⋅ – 0,0332 C x = 24,24 (24.2 on reports and Data Dat a Review screen)
Roche Diagnostics B-38
COBI C D · Version 1.0
cobas c 311 analyzer
3 Photometric principles Prozone 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 on page B-39 Reaction rate method on on page B-42
Antigen readdition 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:
R3
e c n a b r o s b A
Apmp Ap mp 2 R2 S, R1
Ap mp 1 C1 C2 C3
pm p 1
pm p 2
Fig Figure ure B-28
Prozone check - antigen readdition method
C 1 , C 2 , ...
The reaction cell's water blank values (a)
S
Pipetting of sample
R 1 , R 2 , R 3
Pipetting of reagent at R1, R2, and R3 timing
pm p 1 , pm p 2
Prozone measure measure points 1 and 2
Ap mp 1 , Apmp 2
Absorbance at pm p 1 and pm p 2
Time
(a) See Cell Blank Measurement report on on page page B-21 B-21..
Roche Diagnostics COBI C D · Version 1.0
B-39
3 Photometric principles
cobas c 311 analyzer
Prozone check
Programming and calculation
Program a prozone check check on the Analyze tab of the Utility > Application screen according to the following description:
Fig Figure ure B-29
Application parameters of an application with prozone check
To the rright ight of the Prozone Limit field there are nine boxes: [ lower limit ] [ upper limit ] [ pm p 1 ] [ pm p 2 ] [ 0 ] [ 0 ] [ comp. ] [ 0 ] [ 0 ] o
The first two boxes indicate the lower and upper prozone limits (in Abs × 10 4).
o
Thee nex Th nextt fo four ur bo boxxes are are for for th thee p pro rozo zone ne meas measur uree po poin ints ts ( pm p ): O
3rd 3rd entr entry: y: Firs Firstt p prrozon ozonee measu easurre poin points ts ( pm p 1 )
O
4th 4th eent ntry ry:: SSeecond ond pr prozo zone ne meas measur uree p po oin ints ts ( pm p 2 )
O
5th entry: Set to zero for this method
O
6th entry: Set to zero for this method
pm p 2 ≤ 57. If all entries are set to zero, Appropriate values are: 2 ≤ pm p 1 < 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 l ower 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.
Roche Diagnostics B-40
COBI C D · Version 1.0
cobas c 311 analyzer
3 Photometric principles Prozone check
Prozone check value calculation
The calculation of the prozone check value uses the following equation: Equation Equatio n B-17 B-17
P C = A p m p 2 – d ⋅ A pm pmp p 1 with V + V R 1 d = ------------samp ----------------------------------V samp + V R 1 + V R 2
Prozone check value PC Ap mp 2
Absorbance at prozone measure point 2
Ap mp 1
Absorbance at prozone measure point 1
d
Dilution factor
V samp
Sample volume
V R 1
R1 volume
V R 2
R2 volume
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. The following data from the Utility > Application screen screen are used for this example: example:
Prozone check value calculation
T Test est
ALBU2
Assay/Time/Point
[ 2 Point End ] [ 10 ] [ 6 ] [ 15 ] [ 0 ] [ 0 ]
Prozone Limit
[ -32000 ] [ 1000 ] [ 24 ] [ 30 ] [ 0 ] [ 0 ] [ Inside ] [ 0 ] [ 0 ]
The calculation of the prozone check value uses the following equation: Equatio Equ ation n B-1 B-18 8
P C = A p m p 2 – d ⋅ A pm pmp p 1 with V + V R 1 + V R 2 d R 3 = ------------samp ------------------------------------------------V samp + V R 1 + V R 2 + V R 3
Symbol
Definition
PC
Prozone check value
Ap mp 2
Absorbance at prozone measure point 2
0.9951
Ap mp 1
Absorbance at prozone measure point 1
1.1070
d R 3
Dilution factor (correcting for R3 addition)
V samp
Sample volume
6.0 µL
V R 1
R1 volume
100 µL
V R 2
R2 volume
20 µL
V R 3
R3 volume
26 µL
T Table able B-11
Value
Definitions and values for quantities used in the calculation
Roche Diagnostics COBI C D · Version 1.0
B-41
3 Photometric principles
cobas c 311 analyzer
Prozone check
Applying these values to the above formulas (Equati ( Equation on B-18) B-18) yields: 126 ( 6,0 µ L + 100 µ L + 20 µ L ) d R 3 = --------------------------------------------------------------------------------- = --------- = 0,8289 ( 6,0 µ L + 100 µ L + 20 µ L + 26 µ L ) 152
Therefore: P C = A p m p 2 – d ⋅ A p pm m p 1 PC = 0,9951 – 0,8289 ⋅ 1,1070 = 0,0775
The calculated prozone check value is compared to the lower and upper prozone limits on Utility > Application > Analyze. In In the above above calculated example example the 4 prozone check check value is 0.0775 0.0775 × 10 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: v (pmp 3, pmp 4)
∆ A (pm p 4, pmp 3) v (pmp 1, pmp 2) e c n a b r o s b A
R2/R3
∆ A (pm p 2 pm p 1) ,
S, R1
C1 C2 C3
p mp 3 pm p 1 pm p 2 pmp
pm p 4
Time
Fig Figure ure B-30
Prozone check - reaction rate method
C 1 , C 2 , ...
The reaction cell's water blank values (a)
S
Pipetting of sample
R 1
Pipetting of reagent at R1 timing
R 2 , R 3
Pipetting of reagent at R2 or R3 timing
pm p n
Prozone measure measure point n, with n = 1, 2, 3, and 4
v (pmp n , pmp m )
Rate of change in absorbance between pm p n and pm p m
between pm p n and pm p m ∆ A (pm p n , pmp m ) Absorbance difference (a) See Cell Blank Measurement report on on page page B-21 B-21..
Roche Diagnostics B-42
COBI C D · Version 1.0
cobas c 311 analyzer
3 Photometric principles Prozone check
Programming and calculation
To the rright ight of the Prozone Limit field there are nine boxes: [ lower limit ] [ upper limit ] [ pm p 1 ] [ pm p 2 ] [ pm p 3 ] [ pm p 4 ] [ comp. ] [ 0 ] [ 0 ] o
The first two boxes indicate the lower and upper prozone limits (in Abs × 10 4).
o
Thee nex Th nextt fo four ur bo boxxes are are for for th thee p pro rozo zone ne meas measur uree po poin ints ts ( pm p ): O O
3rd 3rd entr entry: y: Firs Firstt pr proz ozon onee measu easurre poin pointt ( pm p 1 ) 4th 4th entr try: y: Sec Second ond p prrozo ozone measu easurre poin pointt ( pm p 2 )
O
5th 5th entry ntry:: Thir Third d pr proz ozon onee measu easurre poin pointt ( pm p 3 )
O
6th 6th entry ntry:: Fou Fourt rth h prozon ozonee measu easurre p poi oint nt ( pm p 4 )
pm p 2 ≤ 57 and 1 ≤ pm p 3 < pm p 4 ≤ 57. If all Appropriate values are: 1 ≤ pm p 1 < 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 l ower 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 × 10 4) for the difference in pm p 2 . If the measured difference between these absorbance between pm p 1 and points falls below the limit, the prozone check is neglected.—In other words: –4
1 < F ×10 If Ap mp 2 – Apmp , reaction rate prozone check is not performed, where F is defined in the eighth box pm p 4 . If the Likewise, the ninth box defines the limit between pm p 3 and measured difference falls below the limit, the prozone check is neglected.—In other words: –4
3 < G ×10 , reaction rate prozone check is not performed, Apmp mp 4 – Apmp If Ap where G is defined in the last box of the Prozone Limit line.
Prozone check value calculation
The calculation of the prozone check value uses the following equation: Equatio Equ ation n B-1 B-19 9
P C = [ v (p m p3 pmp v (pmp 1, pmp 2) ] × 100 with , 4) ⁄ v (pmp 3 pmp Apmp 3 ) ⁄ ( pm p 4 – pm p 3 ) , 4) = ( Apmp 4 – v (pmp 1 pmp Apmp 1 ) ⁄ ( pm p 2 – pm p 1 ) , 2) = ( Apmp 2 –
PC
Prozone check value
pm p n
Prozone measure measure point n (with n = 1, 2, 3, or 4)
v (pmp n , pmp m )
pm p m Rate of change in absorbance between pm p n and
difference between pm p n and pm p m Apmp Ap mp n – Apmp m Absorbance pm p n – pm p m
pm p m Time difference between pm p n and
Roche Diagnostics COBI C D · Version 1.0
B-43
3 Photometric principles
cobas c 311 analyzer
Summary of assay techniques
The calculated calculated proz prozone one check check value ( PC ) is displa displayed yed on the the Reaction Reaction Monitor Monitor window and compared to the range between the lower and upper prozone limits, which is defined in the first two t wo boxes in the Prozone Limit line. Unde Underr the following conditions a prozone data alarm is issued: o
A prozone data alarm is issued if PC lies inside the prozone interval and Inside is displayed in the seventh text box in the Prozone Limit line.
o
Likewise, a prozone data alarm is issued if PC lies outside the prozone interval and Outside is displayed in the seventh text box.
In case of an alarm, the test result is flagged with >Kin on tthe he Reaction Monitor, Monitor, on the Data Review screen, and the prozone data alarm is printed on the patient report.
Summary of assay techniques Assay type
Measure points
Calculation of unknow n
1 Point
1 ≤ mp 1 ≤ 57
2 Point End
1 ≤ mp 1 < mp 2 ≤ 57
C x = [ K ( A x – A b ) + C b ] ⋅ IF A + IF B
2 Point Rate
1 ≤ mp 1 < mp 2 ≤ 57
C x = [ K ( v x – v b ) + C b ] ⋅ IF A + IF B
Rate A with sample blank
1 ≤ mp 3 < mp 4 < mp 1 < mp 2 ≤ 57, mp 3 + 2 < mp 4 , mp 1 + 2 < mp 2
C x = [ K ( v (mp 3, mp 4) – v b ) + C b ] ⋅ IF A + IF B
Rate A
1 ≤ mp 1 < mp 2 ≤ 57, mp 1 + 2 < mp 2
C x = [ K ( v x – v b ) + C b ] ⋅ IF A + IF B with
C x = [ K ( A x – A b ) + C b ] ⋅ IF A + IF B
v x = v (mp 2, mp 1) – d ⋅ v (m p4 , mp 3)
T Table able B-12
Summary of assay techniques
Roche Diagnostics B-44
COBI C D · Version 1.0
cobas c 311 analyzer
3 Photometric principles Summary of assay techniques
Endpoint assay example graphs
Rate assay example graphs
e c n a b r o s b A
e c n a b r o s
S, R1
R2, R3
Am p 1
v x
S, R 1
b A
R 2 /R 3
C1 C1 C2 C2 C3
C1 C2 C2 C3 Time
mp 1
1 Point assay
mp 2 Time
mp 1
Rate A assay
S, R1 v (mp , mp ) 3 4 R2/R3
v (mp 1, mp 2)
R2/R3 e c n a b r o s b A
R1
e c n a b r o s b A
Am p 2
S
Am p 1 C1 C2 C3
mp 1
mp 2
C1 C2 C3
Time
2 Point End assay
mp 3
mp 4
mp 1
mp 2
Time
Rate A with sample blank correction
e c n a b r o s b A
Am p 2 S, R1
R2/R3
Am p 1
C1C2C3
mp 1
mp 2
Time
2 Point Rate assay
T Table able B-13
Reaction time courses for individual assay types
Roche Diagnostics COBI C D · Version 1.0
B-45
3 Photometric principles
cobas c 311 analyzer
Summary of assay techniques
Roche Diagnostics B-46
COBI C D · Version 1.0
cobas c 311 analyzer
4 Serum index principles Table of contents
Serum index principles
This chapter provides you with an overview of the t he serum index test principles used by the cobas c 311 analyzer.
In this chapter
Chapter
Introduction ............................................................ ............................ .............................................................. ................................................. ................... Definition of serum indices ......................................... ......... .............................................................. ............................................ .............. Measurement Measurem ent of serum indices indices ................................................................ .................................. .............................................. ................ Evaluating serum indices ...................................... ...... .............................................................. ................................................... ..................... Serum index data alarms .......................................................... ........................... .............................................................. ...............................
4 B-49 B-49 B-49 B-49 B-51 B-51
Roche Diagnostics COBI C D · Version 1.0
B-47
4 Serum index principles
cobas c 311 analyzer
Table of contents
Roche Diagnostics B-48
COBI C D · Version 1.0
cobas c 311 analyzer
4 Serum index principles Introduction
Introduction A number of diseases result in increased amounts of chromogens such as bilirubin o orr hemoglobin, or lipemic particles, par ticles, which increase the turbidity. turbidit y. These chromogens interfere with many photometric assays. However, this interference can be quantified by means of serum index i ndex measurements. Serum indices are calculations of absorbance measurements that provide a semiquantitative representation of levels of icterus, hemolysis, or lipemia (turbidity) (turbidit y) present in patient samples.
Definition of serum indices The icterus index, I, is reported in icterus units that are linear, linear, up to 60 mg/dL, and semi-quantitative. For example, an icterus index of 20 is equivalent to a known bilirubin concentration concentration of approximately approximately 20 mg/dL. The hemolysis index, H, is reported in hemolysis units that are linear, up to 1000 mg/dL, and semi-quantitative. For example, a hemolysis hemolysis index of 500 is equivalent to a known hemoglobin concentration concentration of approximately 500 mg/dL. The lipemia index, L, is reported in i n lipemia units corresponding to mg/dL of Intralipid® (Kabi-Pharmacia, Inc.), an artificial lipid material. These units are linear, up to 2000 mg/dL, and semi-quantitative. For example, example, an L index of 1000 is equivalent to a 1000 mg/dL Intralipid solution.—Hence, the L index provides provides an estimate of sample’s turbidity, not its concentration of triglycerides.
Measurement of serum indices The analyzer takes an aliquot of the patient pat ient sample, dilutes it wit with h 0.9% NaCl, and then measures the absorbances at three pairs of wavelengths: o
For measurement measurement of lipemia (L), wavelengths 700/660 nm are used because this range is free from influence by hemolysis and icterus (see Figure Figure B-31 B-31 below).
o
Hemolysis (H) is measured at 600/570 nm and correction is made for absorption due to lipemia.
o
Icterus (I) is measured at 505/480 505/480 nm and correction is made for absorption due to lipemia and hemolysis.
Shown below are example absorption spectra of turbid serum, hemolytic solution, and bilirubin solution.
Roche Diagnostics COBI C D · Version 1.0
B-49
4 Serum index principles
cobas c 311 analyzer
Measurement of serum indices
Lipemic serum Bilirubin solution e c n a b r o s b A
Hemolytic solution
340
Fig Figure ure B-31
480 505 54 5 46 570
600 66 6 60 700 Wavelength [nm]
Example absorption spectra of a turbid (lipemic) serum, a hemolytic solution, and a bilirubin solution
Calculation of serum indices
To obtain the serum indices L, H, and I from the sample’s absorbance values, the analyzer uses the following formulas: Equatio Equ ation n B-20
1 Ab s 1 ) L = --- ⋅ ( Abs C
Equatio Equ ation n B-21
bss 1 ) H = -1-- ⋅ ( Ab s 2 – B ⋅ A b A
Equatio Equ ation n B-22
1 bss 2 – F ⋅ A b bss 1 ) I = ---- ⋅ ( Ab s 3 – E ⋅ A b D
L , H , I
Serum indices for lipemia, hemolysis, icterus
C , A , D
Factors for conversion of absorbance values (×104) to serum indices
Ab s 1
Bichromatic absorbance absorbance readings at 700 and 660 nm for lipemia
Ab s 2
Bichromatic absorbance absorbance readings at 600 and 570 nm for hemolysis
Ab s 3
Bichromatic absorbance absorbance readings at 505 and 480 nm for icterus
B
Corrects hemoglobin measurement Ab s 2 for lipemia
E , F
Correct bilirubin measurement Ab s 3 for hemoglobin and for lipemia
C, A, and D are sample dilution-dependent and unit-dependent scaling factors to provide semi-quantitative interference levels. B, E and F are correcting factors which correct overlapping interference spectra. They are independent of sample dilution since they are based on ratios rat ios of absorbances. Serum indices can be programmed in either conventional or SI units. Make sure that the correct scaling factors are set for the units you chose. The units should be the same as those used in test results. e
For more information on programming Serum indices, refer to the Online Help.
Roche Diagnostics B-50
COBI C D · Version 1.0
cobas c 311 analyzer
4 Serum index principles Evaluating serum indices
Evaluating Evalu ating serum indices The results should fall in the following ranges, corresponding to an approximate amount of the chromogen indicated: Serum index
Conventional units
SI units
Lipemia index
L
0-1000 mg/dL
0-11 mmol/L
Intralipid
Hemolysis index
H
0-1000 m mgg/dL
0-620 µ µm mol/L
Hemoglobin
Icterus index
I
0-60 m mgg/dL
0-1000 µm µmol/L
Total bilirubin
T Table able B-14
Once the serum indices are determined, refer to the Limitations section of the application’ss package insert to assess the results. This indicates the index up to which application’ potential interference is within the Roche Diagnostics specification or when the t he hemolyzed, icteric, or lipemic sample may not be used with the respective application. Results which fall outside the permitted range are also flagged.
Serum index data alarms Upper limits for serum indices can be defined individually for each test. Limit values are loaded with the application and displayed in the serum index boxes (L, H, and I) on the Range tab of the Utility > Application screen. If a measured serum index 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. Note that these are just example values! Real values are downloaded or can be retrieved from package inserts.
Roche Diagnostics COBI C D · Version 1.0
B-51
4 Serum index principles
cobas c 311 analyzer
Serum index data alarms
Fig Figure ure B-32
Utility > Application > Range with limits set fo forr all three serum indices
If the obtained L or H index is greater than 10 and/or the I index is ggreater reater 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.
Fig Figure ure B-33
For GLUC2 the limit of 10 for the L index is exceeded. Therefore a >Index data alarm is attached to the result.
Roche Diagnostics B-52
COBI C D · Version 1.0
Calibration
C
5
ISE unit unit - IIon on selec selectiv tivee ele electr ctrod odee cal calib ibra ratio tion n . . . . . . . . . . . . . . . C-3 C-3
6
Phot Photom ometri etricc cal calib ibra ratio tion n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-9
cobas c 311 analyzer
5 IIS S E unit - Ion selective electrode calibration Table of contents
ISE unit - Ion selective electrode calibration
This chapter provides you with an overview of the calibration calibrati on of ion-selective electrode tests used by the cobas c 311 analyzer.
In this chapter
Chapter
5
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
Roche Diagnostics
COBI C D Version 1.0
C 3
5 IIS SE unit - Ion selective electrode calibration Table of contents
Roche Diagnostics
cobas c 311 analyzer
COBI C D Version 1.0
C 4
cobas c 311 analyzer
5 IIS S E unit - Ion selective electrode calibration ISE calibration
ISE calibration The following ISE calibrators are used depending on the t he calibration method: o
Std (1) or S1: ISE Low, a water-based water-based solution
o
Std (2) or S2: ISE High, a water-based water-based solution
o
Std (3) or S3: o o
For global use: ISE Comp., a serum-based solution, is used for full calibrations. For use in US only: ISE High (compensated) with compensated set points is used for full calibrations.
The following table displays all calibration calibr ation methods and the corresponding calibrators. Method
Used ISE calibrators
Blank
Only ISE Com Comp. p. (not recommended in US)
2 Point
ISE Lo Low w and ISE ISE Hig High h
Full
For global global use: ISE ISE Low Low,, ISE High, and and ISE Com Comp. p. For use use in U US S only: ISE Low, IISE SE High, and ISE High (comp (compensated ensated))
T Table able C-1
Used ISE calibrators depending on the calibration method
Additionally, an internal standard (IS) will be measured. The calibration interval for Additionally, all ISE applications is 24 hours.
For each calibration, the standard solutions are aspirated into the electrode cartridges, and—after equilibration occurs at the electrode membrane—the 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.
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Slope 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: E H – E L Equatio Equ ation n C-1
--------S = ------------C H log ⎛ ------⎞ ⎝ C L ⎠
S
Slope
E H
EMF (voltage) of high standard
E L
EMF (voltage) of low standard
C H
Concentration Concen tration of high standard
C L
Concentration Concen tration of low standard
Due to factors such as the condition of the electrodes, the t he measured slope may deviate from this ideal slope. Therefore, the slope obtained should fall within the following fol lowing ranges: Na+
50 to to 68 mV
+
50 to to 68 mV
K
-
Cl
-40 to to -68 mV
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, tthe he 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. Equatio Equ ation n C-2
C IS = C L × 10
( E IS – E L ) ⁄ S
C IS
Concentration of the specific ion in the internal standard
C L
Input concentration of the low standard
E IS
EMF (voltage) of the internal standard for the specific ion
E L
EMF (voltage) of the low standard for the specific ion
S
Slope
The calculated value of the internal standard, as well as the voltage, is shown on the Calibration report.
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5 IIS S E unit - Ion selective electrode calibration One-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 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 aqueous standards and human serum matrix. These differences are very small for regularly maintained ISE units. However, However, under certain conditions these differences may become more prominent, thus requiring compensation. The ISE unit is in an optimal condition when the compensation values (C. Value) are stable and negligible low.
Compensation value calculation The concentration of ions in the compensator is calculated according to the following formula: Equatio Equ ation n C-3
( E C – E IS ) ⁄ S S 3 C o n c = C IS ×10
S 3 Conc Concentration of ions in the ISE Standard 3 (S3) C IS
Concentration of the internal standard, determined during calibration
E C
EMF (voltage) of the compensato compensatorr for the specific ion
E IS
EMF (voltage) of the internal standard for the specific ion
S
Slope
The formula for finding fi nding the compensation value (C. Value): C. Value 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 Utility > Applic Application ation > Calib., a Cal.E alarm alarm is issued.
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Reference 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 referencee point for all measurements. That is, all reported voltages are readings from referenc which the voltage of the reference electrode has been subtracted.
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6 Photometric calibration Table of contents
Photometric calibration
This chapter provides you with an overview of the calibration calibratio n types used by the cobas c 311 analyzer for photometric assays. The K factor, calibration updates and calculating results are also discussed.
In this chapter
Chapter
6
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 Roche Diagnostics
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Table 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 ....................................................................................... C-34 Assay types .......................................................................................................... C-34
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6 Photometric calibration Calibration checks
Calibration checks For each photometric application, the following checks that automatically verify the reliability of calibrations are available. If a check value lies outside the configured check limits, an alarm is issued. This section briefly explains the calibration checks and the associated alarms. Calibration checks
Associated data alarms
SD limit check
SD.E
Duplicate limit check
Dup.E
Sensitivity limit check
Sens.E
S1 Abs. limit check
S1A.E
Std. check
Std.E
T Table able C-2
Calibration checks and associated data alarms
The limits of the calibration checks are configure configured d under Utility Utility > Application > Calib.
Fig Figure ure C-1
SD limit
Calib. tab of the Utility > Application screen
When calibrating nonlinear or multipoint linear tests, the instrument performs the following check: For each calibrator, an absorbance value is calculated from the given concentration and the current calibration curve. This calculated absorbance absor bance is compared to the measured absorbance. If the difference of the two exceeds the SD limit value, an SD.E alarm is issued. The SD limit value is defined in the SD Limit box (in Abs × 10 4). An SD limit value of 999.9 denotes the check will be omitted. In case an SD.E alarm occurs, measurement is still possible and the calibration calibrat ion curve is updated. However, However, trace the cause of the alarm before you proceed to sample measurement. The SD value is printed out together with the result of calibration.
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Calibration checks
Duplicate limit
All photometric calibrators are run in duplicate. The duplicate check calculates the % error and the absolute absorbance error (difference) (difference) between these duplicate measurements. The obtained check check values are compared to the % error limit and the absorbance error limit. The % error limit is defined in the first Duplicat Duplicatee Limit box. The absorbance error limit is defined in the second Duplicate Limit box (Abs.). The DE Abs. are calculated as follows: corresponding check values DE % and Ab s 2 – Abs Ab s 1 Abs. = Ab ----------s ---2----+ - -----Ab --------s --1 -- --)-- ⁄ ----2 - ⋅ 100 and DE Abs. DE % = (- Ab Abs s 2 – Ab s 1 , Ab s 2 denote the two absorbance readings, taken for each calibrator where Ab s 1 and
(duplicate readings). If both the % error and the absorbance error ar aree out of range, a Dup.E alarm is issued indicating a failed calibration. The calibration curve of the affected test is not updated. e
Sensitivity limit
For more details see also Duplicate limit check (Dup.E) on page page D-9 D-9..
Sensitivity, here, refers to the ratio of an absorbance difference to a concentration difference. It is calculated from the t he measured absorbance values and ggiven iven cali co conc ncen entra tratio tion n valu values es of of the the blan blank k cali calibra brato torr ( S 1 ) and and th thee span span calibra brato torr ( S N ): Abss ( S N ) – Abs ( S 1 ) ⁄ Conc ( S N ) – Conc ( S 1 ) Ab
The sensitivity obtained in a calibration calibrat ion must lie within certain limits: The lower limit is defined in the first Sensitivity Limit box. The upper limit is defined in the second Sensitivity Limit box. If the obtained sensitivity is not within these limits, a Sens.E alarm is issued indicating a failed calibration. The calibration curve of the t he affected test is not updated. S1 Abs. limit
This check sets an upper and lower absorbance absorbance limit for the blank calibrator, Std (1). If the absorbance for Std (1) falls outside these limits, the analyzer analyzer issues a S1A.E alarm indicating an erroneous calibration. The calibration curve cur ve of the affected test is not updated. An S1 Abs. Limit minimum of -32000 and maximum of 32000 denotes the check will be omitted. For linear calibrations, the reagent blank is simply the y-intercept of the calibration curve. For all nonlinear calibration types, the reagent blank is the predicted absorbance for an analyte concentration of zero.
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6 Photometric calibration Calibration checks
Std. check
If any of the data alarms listed below occurs in a calibration, an Std.E alarm is issued. The calibration curve of the affected test is not updated. Choose Alarm (global button) to verify which data alarm has occurred. Data alarm
Data alarm
ABS over (absorbance exceeds 3.0)
>Abs
ADC abnormal (analog/digtal converter)
ADC.E
Calculation not possible
Calc.?
Cell blank abnormal
>Cuvet
Duplicate error (difference between 1st and 2nd calibrator measurement)
Dup.E
Linearity abnormal (for rate assays)
>Lin
Prozone error 2 / Kinetic unstable (reaction rate Prozone method)
>Kin
Mixing power low level
React
Reagent short
Reag.S
Sample short
Samp.S
St Stan anda dard rd so solu luti tion on 1 abso absorb rban ancce (S1A (S1Abs bs)) ab abno norm rmal al
S1A. S1A.E E
T Table able C-3
Updated and non-updated calibration data
The table below shows the data output when data alarm is issued during a calibration. If the working curve is not updated, take t ake necessary measures and perform recalibration. Recalibration may also be required depending on the cause of an alarm even if the working curve is updated. Data alarm
Working curve
Saving on hard disk
Display on Alarm screen
SD.E
Updated
Yes
Provided
Dup.E
Not updated
No
Not provided
Sens.E
Not updated
No
Provided
S1A.E
Not updated
No
Not provided
Std.E
Not updated
No
Provided
T Table able C-4
Roche Diagnostics
Data alarms giving rise to an Std.E alarm when occurring in calibration
Data output in case of data alarm during c alibration
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Calibration overview
Calibration overview Calibration types
The term calibration refers to the determination of a valid relation between the measured signal [absorbance or (for rate assays) a rate of change in absorbance] and the concentration of the analyte of interest. The graphical gr aphical representation of such a signal/concentration relation is the calibration curve also referred to as working curve. The analyzer uses different types of mathematical models to describe this relation. These math models are referred to as calibration types.
Calibration methods
Up to six calibrato calibrators—ab rs—abbrevia breviated ted Std (1), Std (2)... Std (6)—can (6)—can be used for a full calibration. However, However, not all of these need to be used in every update of a calibration. Select one of four different calibration methods met hods to define which calibrators are to be used.
Calibration update types
For calibration methods where only one calibrator is remeasured (Blank and Span), there are three possibilities how the calibration curve is corrected. This choice is made by setting the calibration update type. e
Roche Diagnostics
For more information, see: Calibration types on page C-15 Calibration methods on page C-16 Calibration update types on page page C-1 C-199
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6 Photometric calibration Calibration overview
Calibration types Linear calibrations are used for tests when the absorbance readings plotted against calibrator concentrations lie on a straight line. If a linear calibration is based on two calibrator measurements, it is termed linear two-point calibration. If it i t is based on more than two calibrators, it is termed linear multipoint calibration. Nonlinear calibrations are used for tests whose absorbances at different dif ferent concentrations form a nonlinear but reproducible plot. At least three and a maximum of six calibrators are required for calibration. Available Calibration types typ es are RCM, RCM2T1, and RCM2 T2. In addition, there are two calibration types ty pes whose calibration curves are piecewise defined interpolation functions: Spline and Line Graph. The following table provides an overview of all available calibration types. Calibration type
Math model
Linear
y = a + b ⋅ x
RCM
RCM2T1
RCM2T2
Spline
Line Graph
Cross-reference ) y ( . s b A
a – d y = -------------------- + d x c - ⎞⎠ 1 + ⎛⎝-b
) y ( . s b A
sinh z y = a + b ⋅ ----- --------- with z = c ⋅ x + d 1 + z 2
y = a + r ⋅ ( 1 + s ⋅ x ) –2
) y ( . s b A
Piecewise polynomials of higher degree for the interpolation between calibrator data points Polygon of linear interpolations with N – N -1 C N slopes of ( AN – AN -1 ) ⁄ ( C N )
T Table able C-5
) y ( . s b A
) y ( . s b A
) y ( s . b A
Linear calibration on pagee C-2 pag C-222 Conc. (x)
RCM calibration on pagee C-2 pag C-255 Conc. (x)
See RCM2T1 calibration on page C-2 page C-277 Conc. (x)
RCM2T2 calibration on pagee C-2 pag C-299 Conc. (x)
Spline calibration on pagee C-3 pag C-311 Conc. (x)
Line Graph calibration on Conc. (x)
pagee C-3 pag C-333
Overview of calibration types
K factor A K factor is used in the calculation of sample results. Any test requiring more than just a blank during calibration will have its K factor calculated via the measured absorbances of the blank calibrator calibrator Std (1) and the other calibrator(s). e
For more details, see K factor calculation on page C-19 C-19..
A fixed K factor is used for some tests and is derived at the time of system installation. The respective tests have only their blank (baseline) values updated during calibration.
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Calibration overview
Calibration methods A calibration determines the relation between a measured signal and the concentration of an analyte. This relation, however however,, is dependent on various conditions (including lot variations, age of reagents, instrument parameters) and therefore needs to be updated regularly regularly.. A calibration update can be described either as an adjustment of parameters of the calibration curve or as an adjustment of the measured signal (signal correction) to compensate for changed conditions. Both of these descriptions are mathematically equivalent. Calibrations can be updated manually or automatically. A calibration update does not necessarily include all calibrators used in the full calibration calibrat ion of a test. According to the number of calibrators used, calibrations updates are termed one-point or two-point. In case of one-point calibration update, the signal correction is a simple proportional s and s ' denote the signals obtained with the original adjustment: s ' = r ⋅ s , where and the current system, respectively. respectively. In case of a two-point calibration update, the signal correction is linear: s ' = p ⋅ s + q . On cobas c 311 analyzers, there are four methods available to update calibrations: Blank and Span (which are both one-point calibrations), 2 Point, and Full. These calibration methods are listed below along with corresponding calibrators and calibration types. Calibration method
Calibrator(s) needed
Blank
Std (1) calibrator
For cobas c 311 analyzers water is Linear, RCM, RCM2T1, used as blank calibrator calibrator.. RCM2T1, Spline, Line Graph
Span
Std (N) with N > 1
For this method you must use a calibrator other other than Std (1).
2 Point
Std ((11) calibrator and one additional additio nal Std (N), N > 1
Linear, RCM, RCM2T1, RCM2T1
Full
Std ((11), Std (2 ( 2), Std ((33)… Std ((N N) All calibrators specified for the application(a)
RCM, RCM2T1, RCM2T1, Spline, Line Graph
T Table able C-6
Applicable calibration type
Linear, RCM, RCM2T1, RCM2T1
Calibration methods
(a) Displayed on Utility Utility > Application > Oth Others. ers.
In the following we explain these The calibration methods and show how the calibration curvesections, parameters are updated. following par ameters parameters are used throughout: Definition of parameters
S1Abs
Calibration curve parameter displayed displayed in the S1 Abs. column of the (a) Calibration Result window and on Working Information window (b)
K
Calibration curve parameter displayed in the K column
A , B
Calibration curve parameters displayed in the columns A and B
'
A diacritical mark (’) denotes an updated parameter. parameter. For example, B ' is the new new B parameter of th thee ca calibration libration ccurve urve afte afterr the calibration update.
(a) To display this window, window, choose Calibration > Status > Calibra Calibration tion Result. Result. (b) To display this window window,, choose Calibration > Status > Calibratio Calibration n Result > W Working orking Information. Information.
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6 Photometric calibration Calibration overview
Blank calibration
A Blank calibration is a one-point calibration. calibrati on. Tests Tests are calibrated with the Std(1) calibrator only, and the signal correction is a simple proportional adjustment. The calculation method for various applicable calibration types are listed below. Calibration type
S S1 1 Abs.
K
A
B
Linear
S1Abs ' = s b
RCM
S1Abs ' = s b
Previou s value
Previous value
s b B ' = ---------------- ⋅ B S1Abs
RCM2T1
s b S1Abs ' = -------- ⋅ S1Abs s b b
s b K ' = -------- ⋅ K s b b
Previous value
Previous value
RCM2T2
s b S1Abs ' = ------------------------- ⋅ S1Abs K S1Abs +
s b K ' = ------------------------- ⋅ K Previous value S1Abs + K
T Table able C-7
Applicable calibration types for Blank calibration updates
)
s b )
s b b
S1Abs K ' = --------------- ⋅ K s b
)
Mean signal of Std (1) calibrator from current update measurements measurements (absorbance or rate of change in absorbance) Signal value calculated from the (non-updated) RCM2T1 calibration curve for Std (1) calibrato calibratorr s b K ⋅ sinh B ⁄ ( 1 + B 2 ) b = S1Abs + )
Span calibration
A Span calibration is a one-point calibration. T Tests ests are calibrated with only one o ne calibrator, and this has to be a standard solution other than Std (1). The signal correction is a simple proportional adjustment. The calibrator that corresponds to the Span point (entered on Utility > Application > Calib.) is measured and the previously measured calibration curve is corrected for each applicable calibration type as listed below. Calibration type
Linear RCM
S S1 1 Abs.
K
s N S1Abs ' = --------- ⋅ S1Abs N s N s N S1Abs ' = --------- ⋅ S1Abs s N N
s N N K ' = --------- ⋅ K N s N
)
)
A
B
Previous value
Previous value
s N B ' = --------- ⋅ B s N N
Previous value
Previous value
)
RCM2T1
s N S1Abs ' = --------- ⋅ S1Abs s N N
s N K ' = --------- ⋅ K s N N
RCM2T2
s N S1Abs ' = ------------------------- ⋅ S1Abs S1Abs + K
s N K ' = ------------------------- ⋅ K Previous value S1Abs + K
T Table able C-8
Applicable calibration types for Span calibration updates
)
s N )
s N N
Roche Diagnostics
)
)
Mean signal of Std (N) from current update mea measurements surements (absorbance or rate of change in absorbance) Signal value calculated from the (non-updated) calibration curve for the given concentration concentration value of of Std (N)
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Calibration overview )
The calculated signal value S N N is obtained simply by insertion of the given concentration value x of Std (N) into the function of the calibration curve. For a K ) ⋅ x . Likewise for an RCM Linear calibration, for example, s N N = S1Abs + ( 1 ⁄ calibration, )
Equatio Equ ation n C-4
S1Abs – B s N B . N = ------------------------- + ⎛ x ⎞ A 1 + --⎝K ⎠
)
2 Point calibration
Tests are ca calibrat librated ed using Std Std (1) calibrator calibrator and and one calibrator calibrator Std Std (N) with N > 1. For this calibration update, the signal correction is linear: s ' = q + p ⋅ s . The number of the second calibrator Std (N) is displayed in the Span box on Utility > Application > Calib. The calculation method method depends on the calibration type as listed below. Calibration type
S S1 1 Abs.
K
Linear
S1Abs ' = s b
1 K ' = -- ⋅ K p
b + s b
x A --- ⎞⎠ p ⋅ S1Abs ) ⎛⎝K
B
Previou s value
Previous value
B ' = p ⋅ B
Previous value
RCM
S1Abs ' =
RCM2T1
B sinh S1Abs ' = s b – p ⋅ K -------------- 1 + B
K ' = p ⋅ K
Previous value
RCM2T2
S1Abs ' = s b – p ⋅ K
K ' = p ⋅ K
Previous value
T Table able C-9
Applicable calibration types for 2 Point calibration updates
(
b – s b
A
p
Calibration update parameter p = ( s N – s b ) ⁄ ( s N N – s b b )
s b
The currently measured signal (absorbance or rate of change in absorbance) for for Std (1) calibrator
s N
The currently measured signal (absorbance or rate of change in absorbance) for the calibrator Std (N)
)
s b b
)
s N N
)
)
Signal value calculated from the (non-updated) calibration curve for Std (1) calibrator calibrator Signal value calculated from the (non-updated) calibration curve for the given concentration concentration value of of Std (N)
Full calibration
Tests are calibrated using all calibrators specified on Utility > Application > Others. After this calibration, all parameters of the calibration curve are updated. The parameters of a test’s calibration curve are displayed on the Calibration Result window (choose (choose Calibration > Status > Calibration R Result). esult). Parameters Parameters of linear calibration curves are updated by linear regression, and nonlinear calibration curves cur ves are updated using a nonlinear regression algorithm. Applicable calibration types are Linear Li near multipoint (with more than two calibrators), RCM, RCM2T1, RCM2T2, Spline, and Line Graph.
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6 Photometric calibration Calibration overview
Calibration update types If you are updating a calibration using either a Blank update or a Span update you may select how how the calibration is updated from Utility > Application > Calib. using the Update Type box. The following calibration types can be updated by either the Blank or Span method and may use this update feature: RCM, RCM2T1, RCM2T2, Spline, and Line Graph. The calibrator used is either either Std (1), for a Blank calibration, or it is defined defined in the Span box on Utility > Application > Calib. > Calibration Type Type area. There are three update types available: None, Difference, and Ratio. None
If None is chosen as the entry in the Update Type Type box, then neither Difference nor Ratio calibration update values are applied. a pplied. The calibration update occurs as described for either a blank or span calibration, depending on the calibration type chosen.
Difference
The absorbance difference to the previous calibration is measured for the one defined calibrator only. This difference is then added to each of the test’s test’s standard absorbance values. This moves the calibration curve up or down, maintaining its original slope.
Ratio
The test’s absorbance value is measured with the one defined calibrator only. The ratio of this value to the previous yields an adjustment factor. Each of the test’s standard absorbance values is then multiplied by this factor. This adjusts the slope of the calibration curve, maintaining its original origi nal y-intercept.
K factor calculation This section shows how K factors are calculated from absorbance and concentration values for tests that are based on linear two-point calibration curves. Tw Two o examples are given: One for an endpoint assay and one for a rate assay. assay. After a successful calibration, an updated S1 Abs. value is shown on both the W Working orking Information window and the first column of the S1 on the t he Calibration Monitor report.
Fig Figure ure C-2
Working Information window
The absorbance value (or rate of change in absorbance) of the second calibrator is printed in the first column under S2 on the Calibration Monitor report. Roche Diagnostics
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Calibration overview
These new values are used to calculate the K factor. When displayed on the Working Working Information window, window, the K factor is automatically rounded and multiplied by the correct power of 10, according according to the decimal placement placement of the Std (1) concentration. Endpoint assay example
The formula for endpoint assays is: Equatio Equ ation n C-5
K = ( C N – C b ) ⁄ ( A N – A b )
C b
Concentration Concen tration value for Std (1)/blank calibrator
C N
Concen Con centration tration va value lue for the sec second ond cal calibrat ibrator or Std (N), N > 1
A b
Absorbance of Std (1)/blank calibrator (S1 Abs.)
A N
Absorbanc Abso rbancee of the sec second ond ca calibrat librator or Std (N), N > 1
A glucose test is calibrated calibrated with water as Std (1) and a secon second d calibrator, calibrator, Std (2), with a glucose concentration of 10.8 mmol/L. Mean values of the measured absorbance values are 0.0036 for Std (1) and 0.8739 0.8739 for Std (2). The K factor is calculated as follows: Equatio Equ ation n C-6
K = ( 10,8 – 0,00 ) ⁄ ( 0,8 ,87 739 – 0,0036 ) K = 10 10,8 ,8 ⁄ 0,87 0,8703 03 = 12,41
This K factor can now be used to calculate results from test absorbances. e
Rate assay example
For more information on calculation of endpoint assay results, see: Example of a 2 Point End assay on on page B-12 Calculation of concentration on page page B-1 B-155
The formula for rate assays is: Equatio Equ ation n C-7
K = ( C N – C b ) ⁄ ( v N – v b )
C b
Concentration Concen tration value for Std (1)/blank calibrator
C N
Concen Con centration tration va value lue for the sec second ond cal calibrat ibrator or Std (N), N > 1
v b
Rate of change in absorbance of the reaction with Std (1)/blank calibrator
v N
Rate of change in absorbance of the reaction with the calibrator Std(N), td(N), N> 1
An AST (aspartate aminotransferase) test is calibrated calibrated with water as Std (1) and a second calibrator with a concentration of 94.2 U/L. Mean values of the measured rates of change in absorbance are v b = –0,0006 for Std (1) and v x = –0,01575 for the second calibrator. The K factor is calculated as follows: Equatio Equ ation n C-8
K = ( 94,2 – 0,0 ) ⁄ [ – 0,0486 – ( –0,0006 ) ] K = 94,2 ⁄ ( –0,0480 ) = – 1962,5
This K factor can now be used to calculate results from test absorbance rates. e
Roche Diagnostics
For more information on calculation of rate assay results, see: s ee: Example of a Rate A assay on on page B-17 Result calculation on page B-19
COBI C D · Version 1.0
C-20
cobas c 311 analyzer
6 Photometric calibration Calibration overview
Introduction to weighting It is possible to apply a weighting function during the curve fitting process that favors those calibrator points with a lower absorbance (or rate of change in absorbance). This may result in a more accurate curve fit in that particular concentration range. Calculation without weighting
When weighting is not used (e (entry ntry of 0 in the Weight Weight field on Utility > Application > Calib.), the curve fit is optimized by varying the parameters of the calibration function to minimize the sum of the t he residuals. The residuals are the squares of the differences between the actual absorbance values for each calibrator and the absorbance calculated from the calibration function. n
Equatio Equ ation n C-9
∑ [ Ai – f (C i ) ]
2
→ min
i = 1
A i
Actual absorbance (or rate of change in absorbance) of calibrator i
f(C i )
Absorbance (or rate of ch change ange of absorbance) o off calibrato calibratorr i calculated by the calib calibration ration func function tion from its con concen centratio tration n ( C i )
i = 1 … n
Numbers of calibrators used
Calculation with weighting
When weighting weighting is used (entry (entry of 1 or 2 in the Weight field on Utility > Application > Calib.), each of the residuals is multiplied by a weight factor during the curve fitting process thus: n
Equatio Equ ation n C-1 C-10 0
∑ { w i [ Ai – f (C i ) ] }2 → min i = 1
w i
Weight factor for calibrator point i
All other symbols
As described above
Weighting Weigh ting factors factors
The weighting factor is inversely related to the absorbance of the calibrator, so that those calibrator points with a lower absorbance will have a larger weighting factor.
Roche Diagnostics
o
If an entry of 1 is made in the Weight field, then the weighting factor for calibrator point i is: w i = 1 ⁄ [ g(A i ) ] , where g(A i ) is a function of the absorbance (or rate of change in absorbance) of calibrator i .
o
If an entry of 2 is made in the Weight field, then the weighting factor for calibrator point i is: w i = 1 ⁄ [ g(A i ) ]2 .
COBI C D · Version 1.0
C-21
6 Photometric calibration
cobas c 311 analyzer
Linear calibration
Linear calibration Water is commonly used as a zero or blank calibrator. For Linear 2 point calibration, the absorbance of water and a second calibrator is measured. These two points are used to establish a linear plot, and its slope is used in the calculation of subsequent control and patient results. Paramete Para meters rs on Utility > Application Application > Calib. Calib.:: Calib Type
Linear
Point
2
Weight
0, 1, 2
Span Point
2 to 6 (for 2 Point use 2)
Linear two-point calibration graph
A S 2
e c n a b r o s b A
A x
A b
C b
C x
C S 2
When C b = 0 Fig Figure ure C-3
Roche Diagnostics
Linear 2 point calibration graph - Cb = 0
Concentration
COBI C D · Version 1.0
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cobas c 311 analyzer
6 Photometric calibration Linear calibration
A S 2
e c n a b r o s b A
A x
A b
C b
C x
C S 2
Concentration
When C b ≠ 0 Fig Figure ure C-4
Linear 2 point calibration graph - Cb ≠ 0
A x
Sample absorbance value
A b
Absorbance of Std (1)/blank calibrator (S1 Abs.)
A S 2
Absorbance of Std (2)
C b
Concentration Concen tration value for Std (1)/blank calibrator
C x
Concentration Concen tration of the analyte in the ssample ample
C S 2
Concentration Concen tration value for Std (2)
Linear two-point calculation The math model for a Linear calibration is the equation for a straight line y = a + b ⋅ x , where a is the y-intercept and b is the slope. For our purpose, we interpret this equation’s equation’s variables in as follows:
Slope
Roche Diagnostics
x = C
Concentration Concen tration of the analyte
y = A
Absorbance (or rate of change in absorbance for rate assays)
a
Absorbance when the concentration concentration of the analyte is 0
b
Ratio of the change in absorbance to the change in concentration
The slope of a straight line can be derived either by the formula b = ( ∆y ) ⁄ ( ∆x ) (when two points are used) or by the least squares method (when multiple points are used). For the first case, comparison with Figure Figure C-4 C-4 shows that ∆ y = A S 2 – Ab and ∆ x = C S 2 – C b . The formula for the slope can then be solved to b = ( A S 2 – Ab ) ⁄ ( C S 2 – C b ) . This equation shows that b is eq equal ual to to the reciprocal K factor defined defined earlier. earlier. Therefore, b = 1 ⁄ K .
COBI C D · Version 1.0
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6 Photometric calibration
cobas c 311 analyzer
Linear calibration
y-intercept
b ) , where Ab Comparison with Fig Figure ure C-4 C-4 shows that the y-intercept a = A b – ( b ⋅ C is the absorbance and C b the concentration value for Std (1)/blank calibrator.
With slope and y-intercept thus determined, it is now possible to solve the equation y = a + b ⋅ x to x , to calculate the analyte concentration in a patient sample C x : Equatio Equ ation n C-1 C-11 1
y = a + b ⋅ x yields x = -1- ( y – a ) , where b a = Ab – ( b ⋅ C b )
b = 1 ⁄ K
x = C x
y = A x
following equation is obtained: By substitution of a , b , x , and y the Equatio Equ ation n C-1 C-12 2
C x = K [ A x – ( Ab – b ⋅ C b ) ] which is equivalent to C x = [K ( A x – Ab ) + C b ]
Two additional constants are applied to this formula fo rmula to correct the result for systematic bias deriving from the instrument. The final formula for calculation of the concentration is Equatio Equ ation n C-1 C-13 3
C x = [ K ( A x – Ab ) + C b ] ⋅ IF A + IF B
C
Concentration Concen tration of the analyte in the ssample ample
x
K
K factor
A x
Sample absorbance value
A b
Absorbance of Std (1)/blank calibrator (S1 Abs.)
C b
Concentration Concen tration value for Std (1)/blank calibrator
IF A , IF B
Instrument constants representing representing a slope of 1 and an intercept of 0
Assay types Linear 2 Point calibration can be used with the following assay types:
Roche Diagnostics
o o
1 Point assay 2 Point End assay
o
2 Point Rate assay
o
Rate A assay
COBI C D · Version 1.0
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cobas c 311 analyzer
6 Photometric calibration RCM calibration
RCM calibration The RCM calibration applies a working curve in which the absorbance absorba nce increases or decreases in a nonlinear manner as the concentration increases. Paramete Para meters rs on Utility > Application Application > Calib. Calib.:: Calib Type
RCM
Point
2 to 6
Weight
0,1,2
Span Point
2 to 6
RCM calibration gr graph aph
A N e c
A X
n a b A S 3 r o s b A A S 2
A b
C b
C S 2
C S 3
C X
C N
Concentration
Roche Diagnostics
Fig Figure ure C-5
Nonlinear RCM calibration graph
A x
Sample absorbance value
A b
Absorbance of Std (1)/blank calibrator (S1 Abs.)
A S 2 , A S 3 , ...
Absorbance value for Std (2) to Std (6)
A N
Absorbance of Std (N)
C x
Concentration Concen tration of the analyte in the ssample ample
C b
Concentration Concen tration value for Std (1)/blank calibrator
C S 2 , C S 3 , ...
Concentration Concen tration value for Std (2) to Std (6)
C N
Concentration Concen tration value for Std (N)
COBI C D · Version 1.0
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6 Photometric calibration
cobas c 311 analyzer
RCM calibration
RCM calculation The math model for the RCM calibration curve approximation is shown below: Equatio Equ ation n C-1 C-14 4
a – d A = --------------------- + d C c 1 + ⎛--- ⎞ ⎝ b ⎠
A
Absorbance (or rate of change in absorbance for rate assays)
C
Concentration Concen tration of the analyte
a
the absorb Paramete Para meterr re represe presenting nting absorbance ance at zzero ero con concen centratio tration n ( Ab ).
b
Parameter representing representing the conce concentration ntration where the absorbance or Ab . absorbance rate is ½ of the span between Ain f and
c
Parameter describing the curvature of the calibration curve.
d
Parameter representing representing the predicted absorbance or absorbance rate for in infi fin nit itee concen ncentr traatio tion ( A in f ).
The above calibration curve parameters correspond to the values on the Working Information window as follows (to display this window, select Calibration > Status > Calibration Result > Working Information): S1 Abs. column displays parameter a . K column displays parameter b . A column displays parameter c . B column displays parameter d . The formula for sample concentration calculation is shown below: Equatio Equ ation n C-1 C-15 5
C x = ( C + C b ) ⋅ IF A + IF B with a – A x 1 ⁄ c C = b ⋅ ⎛ ---------------⎞ ⎝ A x – d ⎠
C x
Concentration Concen tration of the analyte in the ssample ample
C b
Concentration Concen tration value for Std (1)/blank calibrator
C IF A , IF B
Concentration value before instrument constants adjustment Concentration Instrument constants representing representing a slope of 1 and an intercept of 0
A x
Sample absorbance value
a, b, c, d
Calibration curve parameters as in Equati Equation on C-14
Assay types Nonlinear RCM calibration can be used with the following assay types: t ypes:
Roche Diagnostics
o
1 Point assay
o
2 Point End assay
o
2 Point Rate assay
o
Rate A assay
COBI C D · Version 1.0
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cobas c 311 analyzer
6 Photometric calibration RCM2T1 calibration
RCM2T1 calibration The RCM2T1 calibration applies a working curve in which the absorbance increases in a nonlinear manner as the concentration increases. Paramete Para meters rs on Utility > Application Application > Calib. Calib.:: Calib Type
RCM2T1
Point
2 to 6
Weight
0,1,2
Span Point
2 to 6
RCM2T1 calibration graph
A N e c
A X
n a A S 3 b r o s b A A S 2
A b
C b
C S 2
C S 3
C X
C N
Concentration
Roche Diagnostics
Fig Figure ure C-6
Nonlinear RCM2T1 calibration graph
A x
Sample absorbance value
A b
Absorbance of Std (1)/blank calibrator (S1 Abs.)
A S 2 , A S 3 , ...
Absorbance value for Std (2) to Std (6)
A N
Absorbance of Std (N)
C x
Concentration Concen tration of the analyte in the ssample ample
C b
Concentration Concen tration value for Std (1)/blank calibrator
C S 2 , C S 3 , ...
Concentration Concen tration value for Std (2) to Std (6)
C N
Concentration Concen tration value for Std (N)
COBI C D · Version 1.0
C-27
6 Photometric calibration
cobas c 311 analyzer
RCM2T1 calibration
RCM2T1 calculation The math model for the RCM2T1 calibration curve approximation is shown below: Equatio Equ ation n C-1 C-16 6
z sinh A = a + b ⋅ ---- ---------- with z = c ⋅ C + d 1+ z 2
A
Absorbance (or rate of change in absorbance for rate assays)
C
Concentration Concen tration of the analyte
a, b, c, d
Calibration curve parameters determined by nonlinear regression algorithm
The above calibration curve parameters correspond to the values on the Working Information window window as follows (to display this this window, window, select Calibration > Status > Calibration Result > Working Working Information): S1 Abs. column displays parameter a . K column displays parameter b . A column displays parameter c . B column displays parameter d . The model function for RCM2T1 (Equati (Equation on C-16) C-16) cannot be inverted analytically. 2
n + 1 = ar n ) ] can be used to solve the ar c sinh [ y ⋅ ( 1 + z n However, the iteration series z n However, equation y = sinh z ⁄ ( 1 + z 2) . Thus, the formula for the sample concentration is as follows:
Equation Equatio n C-17 C-17
C x = ( C + C b ) ⋅ IF A + IF B where C is calculated by iteration.
C x
Concentration Concen tration of the analyte in the ssample ample
C b
Concentration Concen tration value for Std (1)/blank calibrator
C
Concentration Concen tration value before instrument constants adjustment
IF A , IF B
Instrument constants representing representing a slope of 1 and an intercept of 0
Assay types Nonlinear RCM2T1 calibration can be used with the following assay types: ty pes: o
1 Point assay
o
2 Point End assay
o
2 Point Rate assay
o
Rate A assay
Roche Diagnostics COBI C D · Version 1.0
C-28
cobas c 311 analyzer
6 Photometric calibration RCM2T2 calibration
RCM2T2 calibration The RCM2T2 calibration applies a working curve in which the absorbance decreases in a nonlinear manner as the concentration increases. Paramete Para meters rs on Utility > Application Application > Calib. Calib.:: Calib Type
RCM2T2
Point
2 to 6
Weight
0,1,2
Span Point
2 to 6
RCM2T2 calibration graph
A b e c A
S 2 n a b r A o X s b A A S 3
A N
C b C S 2
C X
C S 3
C N
Concentration Fig Figure ure C-7
Nonlinear RCM2T2 calibration graph
A x
Sample absorbance value
A b
Absorbance of Std (1)/blank calibrator (S1 Abs.)
A S 2 , A S 3 , ...
Absorbance value for Std (2) to Std (6)
A N
Absorbance of Std (N)
C x
Concentration Concen tration of the analyte in the ssample ample
C b
Concentration Concen tration value for Std (1)/blank calibrator
C S 2 , C S 3 , ...
Concentration Concen tration value for Std (2) to Std (6)
C N
Concentration Concen tration value for Std (N)
Roche Diagnostics COBI C D · Version 1.0
C-29
6 Photometric calibration
cobas c 311 analyzer
RCM2T2 calibration
RCM2T2 calculation The math model for the RCM2T2 calibration curve approximation is shown below: Equatio Equ ation n C-1 C-18 8
A = a + r ⋅ ( 1 + s ⋅C ) – 2
A
Absorbance (or rate of change in absorbance for rate assays) Concentration Concen tration of the analyte
C a , r , s
Calibration curve parameters determined by nonlinear regression algorithm
The above calibration curve parameters correspond to the values on the Working Information window window as follows (to display this this window, window, select Calibration > Status > Calibration Result > Working Working Information): S1 Abs. column displays parameter a . K column displays parameter r . A column displays parameter s . The formula for sample concentration calculation is shown below: Equatio Equ ation n C-1 C-19 9
C x = ( C + C b ) ⋅ IF A + IF B where 1 r C = -- ⋅ ⎛ -------------- – 1⎞ ⎠ s ⎝ A x – a
C x
Concentration Concen tration of the analyte in the ssample ample
C b
Concentration Concen tration value for Std (1)/blank calibrator
C
Concentration Concen tration value before instrument constants adjustment
IF A , IF B
Instrument constants representing representing a slope of 1 and an intercept of 0
A x
Sample absorbance value
a , r , s
Calibration curve parameters as in Equati Equation on C-18
Assay types Nonlinear RCM2T2 calibration can be used with the following assay types: ty pes: o
1 Point assay
o
2 Point End assay
o
2 Point Rate assay
o
Rate A assay
Roche Diagnostics COBI C D · Version 1.0
C-30
cobas c 311 analyzer
6 Photometric calibration Spline calibration
Spline calibration When this calibration type is applied, the ranges between the data points of the measured calibrators are approximated by third degree polynomials so that a smooth calibration curve is obtained. Paramete Para meters rs on Utility > Application Application > Calib. Calib.:: Calib Type
Spline
Point
3 to 6
Weight
0,1,2
Span Point
2 to 6
Spline calibration graph
A N e AN -1 c n a A X b r o s b A
A S 3 A S 2 A b C b
C S 2 C S 3
C X C N --11
C N
Concentration Fig Figure ure C-8
Nonlinear spline calibration graph
A x
Sample absorbance value
A b
Absorbance of Std (1)/blank calibrator (S1 Abs.)
A S 2 , A S 3 , ... A N
Absorbance of Std (2), Std (3), ...Std (N)
C x
Concentration Concen tration of the analyte in the ssample ample
C b
Concentration Concen tration value for Std (1)/blank calibrator
C S 2 , C S 3 , ... C N
Concentration Concen tration value for Std (2), Std (3), ...Std (N)
Roche Diagnostics COBI C D · Version 1.0
C-31
6 Photometric calibration
cobas c 311 analyzer
Spline calibration
Spline calculation In the math model of a spline calibration, data points of calibrators are taken as supporting points for the determination of interpolating functions. The simplest interpolating functions are used in linear interpolation, i nterpolation, where adjacent data points are connected by a straight line. This method is i s used for Line Graph calibrations. e
See Line Graph calibration on page C-33. C-33.
In contrast to the angular polygon of a Line Graph calibration, a smooth curve will result when using piecewise polynomials of a higher degree for the interpolation. i nterpolation. The routine applied for Spline calibrations determines a smooth smoot h cubic spline approximation using third degree polynomials.
Assay types Nonlinear spline calibration can be used with the fo following llowing assay types: o
1 Point assay
o
2 Point End assay
o
2 Point Rate assay
o
Rate A assay
Roche Diagnostics COBI C D · Version 1.0
C-32
cobas c 311 analyzer
6 Photometric calibration Line Graph calibration
Line Graph calibration When this calibration type is applied, the ranges between the data points of the measured calibrators are approximated by linear interpolation. An angular polygon is obtained as calibration curve. Paramete Para meters rs on Utility > Application Application > Calib. Calib Type
Line Graph
Point
3 to 6
Weight
0,1,2
Span Point
2 to 6
Line Graph calibration graph AN A N --11
AS 4 e c n a b r o s b A
A x
AS 3
AS 2 A b C b
C S 2
C S 3 C x
C S 4
C N --11
Concentration Fig Figure ure C-9
Nonlinear line calibration graph
A x
Sample absorbance value
A b
Absorbance of Std (1)/blank calibrator (S1 Abs.)
A S 2 , A S 3 , ... A N
Absorbance of Std (2), Std(3), ...Std (N)
C x
Concentration Concen tration of the analyte in the ssample ample
C b
Concentration Concen tration value for Std (1)/blank calibrator
C S 2 , C S 3 , ... C N
Concentration Concen tration value for Std (2), Std (3), ...Std(N)
C N
Roche Diagnostics COBI C D · Version 1.0
C-33
6 Photometric calibration
cobas c 311 analyzer
Line Graph calibration
Line Graph calculation The math model for Line Graph calibration calibrat ion curve approximation is shown below: Equatio Equ ation n C-2 C-20 0
C N – C N -1 K N -1 -1 = ----------------------- AN – AN -1
K N -1
C N , or A N --11 and Calibration factor for the interval between C N --11 and A N , respectively
A N
Absorbance of Std (N)
A N --11
Absorbance of Std (N-1)
C N
Concentration Concen tration value for Std (N)
C N -1
Concentration Concen tration value for Std (N-1)
The formula for sample concentration is shown below: Equatio Equ ation n C-2 C-21 1
C x = [ K N -1 ( A x – AN -1 ) + C N -1 ] ⋅ IF A + IF B for A x ∈ [A N --11 A , N ]
C x
Concentration Concen tration of the analyte in the ssample ample
A x
Sample absorbance value
IF A , IF B
Instrument constants representing representing a slope of 1 and an intercept of 0
All other symbols
See legend above.
For a calibration based upon N standard solutions—Std (1) to Std (N)—there are are N - 1 calibration curve intervals. The sample absorbance value (or rate of chang changee in absorbance for rate assays) A x determines which of the calibration curve intervals and which of the calibration factors is relevant for the calculation of C x . If A x lies A N the relevant calibration between A N --11 and factor is K N -1 .
Assay types Nonlinear Line Graph calibration can be used with the following assay types: o 1 Point assay o
2 Point End assay
o
2 Point Rate assay
o
Rate A
Roche Diagnostics COBI C D · Version 1.0
C-34
Calculating data alarms
7
D
Calc Calcul ulat atin ingg d dat ataa al alar arms ms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-3
cobas c 311 analyzer
7 Calculating data alarms Table of contents
Calculating data alarms
This chapter provides you with an overview of how some important import ant data alarms are calculated by the cobas c 311 analyzer.
In this chapter
Chapter
7
Introduction ............................................................................................................... D-5 Prozone effect ............................................................................................................. D-5 Linearity verification (>Lin) ...................................................................................... D-7 Sensitivity limit check (Sens.E) ............................................................ ............................... .................................................. ..................... D-9 Duplicate limit check (Dup.E) ................................................................................... D-9 Technical limit check (>T (>Test) est) .......... ......................................... ............................................................ .......................................... ............. D-11 Repeat limit check (>Rept) ...................................................................................... D-12 Reaction limit check (>React) ................................................................................. D-12
Roche Diagnostics COBI C D · Version 1.0
D-3
7 Calculating data alarms Table of contents
cobas c 311 analyzer
Roche Diagnostics COBI C D · Version 1.0
D-4
cobas c 311 analyzer
7 Calculating data alarms Introduction
Introduction Several methods are used by the analyzer to ensure that final f inal results are valid. Data alarms appear on the results printout to indicate possible data errors and are sent to Host. Some of these also activate the audible alarm and initiate the display of the alarm indicator on the global Alarm button.
Prozone effect Some homogenous immunoassays use the principle of antigen/antibody complex formation (agglutination or precipitation) as a measurement techniqu technique. e. The turbidity caused by this specific agglutination or precipitation can be measured by photometric means. The antigen/antibody complex formation is predictable as long as an excess of reagent (antibody) exists. However, However, in patient samples wit with h very high levels of antigen, the reaction may begin to reverse (deagglutination) because of the effect of the excess antigen. This is called a prozone effect. Without checking for this phenomenon, abnormally high levels of antigen in samples may give incorrect or even false normal results. There are two prozone check methods available: Antigen readdition method and reaction rate method. Both of these methods can be applied to any type t ype of assay. Antigen readdition
The analyzer may perform a check for the prozone effect by adding a dilution of the antigen as an additional reagent (R2 or R3). If the reaction continues in the same direction (increasing or decreasing absorbance) as in the initial reaction, then an excess of reagent (antibody) still exists—prozone effect is not occurring. If the t he reaction proceeds in the opposite direction, after additional antigen is added, then prozone effect is occurring and the result is i s invalid. The corresponding data alarm is printed on the patient report. The antigen readdition method is applied when two prozone measuring points are defined on Utility > Application > Analyze ( [ pm p 1 ] [ pm p 2 ] [ 0 ] [ 0 ] ). ).
Roche Diagnostics COBI C D · Version 1.0
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7 Calculating data alarms
cobas c 311 analyzer
Prozone effect
R3
e c n a b r o s b A
Ap mp 2 R2 S, R1
Ap mp 1 C1 C2 C3
Fig Figure ure D-1
Prozone check - antigen readdition method
C 1 , C 2 , ...
The reaction cell's water blank values (a)
S
Pipetting of sample
R 1 , R 2 , R 3
Pipetting of reagent at R1, R2, and R3 timing
pm p 1 , pm p 2
Prozone measuring points 1 and 2 Absorbance at pm p 1 and pm p 2
Ap mp 1 , Apmp 2
Time
pm p 2
pm p 1
(a) See Chapter 3 Photometric principles: Cell Blank Measurement report on on page page B-21 B-21..
Reaction rate method
The reaction rate method verifies the existence of an excess of reagent (antibody) not by repeated addition of antigen but by observation of the reaction rate in the course of the reaction. This method is applied when four prozone measuring points are defined on Utility > Application > Analyze ( [ pm p 1 ] [ pm p 2 ] [ pm p 3 ] [ pm p 4 ] ). ). v (pmp 3, pmp 4)
∆ A (pm p 4, pmp 3) v (pmp 1, pmp 2) e c n a b r o s b A
R2/R3
∆ A (pm p pm p ) , 2
1
S, R1
C1 C2 C3
pm p 1 pm p 2 p mp 3 pm p 4
Time
Fig Figure ure D-2
Prozone check - reaction rate method
v (pmp 1 pmp , 2)
pm p 2 . Rate of change in absorbance between pm p 1 and
All other symbols
See Figu Figure re D-1 D-1 above. above.
e
For more information, see Chapter 3 Photometric principles.
Roche Diagnostics COBI C D · Version 1.0
D-6
cobas c 311 analyzer
7 Calculating data alarms Linearity verification (>Lin)
Linearity verification (>Lin) To verify the linearit linearityy of a rate reaction (rate of change in absorbance), absorba nce), the percentage of nonlinearity is calculated. This values must be less than the Linearity Linearit y Limit, defined on the Utility > System > Application. If the calculated value is above the limit, a > >Lin Lin data alarm is issued. If any absorbance reading taken during the programmed interval exceeds the Abs. Limit parameter, that absorbance reading is excluded from the least squares rate calculation. Depending on the number of measure points of an application, the analyzer calculates the nonlinearity in one of the following ways: o
If there are less than 5 measure points, linearity check is not performed.
o
If there are 5 to 13 measure points, two times three t hree points are used in the calculation.
o
If there are 14 or more measure points, two times six points are used in the calculation.
5-13 measure points
∆ v f e c n a b r o s b A
S, R1
v x
R2/R3 v i
∆
mp 1
mp 2
Time
Fig Figure ure D-3
Linearity verification (5-13 measure points)
S
Pipetting of sample
R1, R2/R3
Pipetting of reagent at R1 timing, and at R2 or R3 timing
mp 1
First photometric measure point
mp 2
Last photometric measure point
v x
Rate of change in absorbance calculated for all measure points between mp 1 and mp 2 by least squares analysis
v i
Rate of change in absorbance calculated for the initial five measure points
v f
Rate of change in absorbance calculated for the final five measure points
The percentage of nonlinearity is the difference between the slope of the initial part of the curve and the slope of the final part of the curve scaled to the overall slope. An LL 1 is the value of the first box in alarm is issued, if [ ( v i – v f ) ⁄ ( v x ) ] ⋅ 100 > LL 1 , where the Linearity Linearity Limit line on Utili Utility ty > Application Application > Analyze. Analyze.
Roche Diagnostics COBI C D · Version 1.0
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7 Calculating data alarms
cobas c 311 analyzer
Linearity verification (>Lin)
14 or more measure points
In principle, the percentage of nonlinearity for 14 or more measure points is calculated in the same way as for 5 to 13 measure points. The only diff difference erence is that v i and v f are calculated on the basis of the initial and final six measure measure points, respectively. LL 2 is the value of the second An alarm is issued, if [ ( v i – v f ) ⁄ ( v x ) ] ⋅ 100 > LL 2 , where box in the Linearity Linearity Limit line on Utility Utility > Application Application > Analyze Analyze..
∆ v f
e c n a b r o s b A
S, R1
v x
R2/R3 ∆ v i
mp 1
Additional conditions for the linearity check
mp 2
Time
Fig Figure ure D-4
Linearity verification (14 or more measure points)
S
Pipetting of sample
R1, R2/R3
Pipetting of reagent at R1 timing, and at R2 or R3 timing
mp 1
First photometric measure point
mp 2
Last photometric measure point
v x
Rate of change in absorbance calculated for all measure points between mp 1 and mp 2 by least squares analysis
v i
Rate of change in absorbance calculated for the initial eleven measure points
v f
Rate of change in absorbance calculated for the final eleven measure points
To the right of the Linearity Limit field on the Utility > System > Application there are four boxes: Line Lineari arity ty Li Limi mitt [ limi limitt 5-13 5-13 mp ]% [ lim limit it ≥ 14 mp]% mp]% [ co cond ndit itio ion n 1 ] [ con ondi diti tion on 2 ] o
The first two boxes boxes indicate the linearity limits (in Abs × 104/min) for 5 to 13 and 14 or more measure points, respectively.
o
The third and forth boxes define additional conditions for the linearity check. The entry in the third box defines a minimum rate of change in absorbance (allover (allov er slope in Abs Abs × 104/min) for v x . If the measured rate falls below this minimum, the linearity check is neglected.—That is, the third box defines the variable T for the following condition: O
–4
If v x < T ×10 , linearity check is not performed.
v f (in The entry in the fourth four th box defines a minimum difference between v i and 4 Abs Abs × 10 /min). If the measured difference v i – v f falls below this minimum, the
linearity check check is neglected.—That is, the fourth box box defines the variable D for the following condition: O
–4
If v i – v f < D ×10 , linearity check is not performed.
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7 Calculating data alarms Sensitivity limit check (Sens.E)
Sensitivity limit check (Sens.E) An upper and and lower sensitivity sensitivity limit is designated on Utility > Application > Calib. for each photometric application. These values relate to the minimum and maximum absorbance changes which must be satisfied between the blank and span calibrators during calibration. The sensitivity observed during calibration is calculated as follows: Equatio Equ ation n D-1
Abss ( S N ) – Abs ( S 1 ) Ab ----------------------------------------------------------Conc ( S N ) – Conc ( S 1 )
where S N is the span calibrator and S 1 is the blank. If the sensitivity observed is not within the sensitivity limits, a Sens.E alarm is issued indicating a failed calibration. All calibrations that affect the factor setting for the test will be error checked against the sensitivity limit calculated from the span calibrator.
Duplicate limit check (Dup.E) A duplicate limit for calibrator acceptability acceptability is designated on Utility > Application > Calib. The entry in the first f irst Duplicate Limit text box defines the % error limit. The entry in the second box defines the absorbance error limit. The corresponding check values are calculated as follows: Ab s 2 – Abs Ab s 1 DE % = ------------------------------- -------------- ⋅ 100 ; DE Abs. = Ab s 2 – Ab s 1 ( Ab s 2 + Ab s 1 ) ⁄ 2 DE %
Relative duplicate error: Calculated value for the % error of a calibrator’ calibrator’ss absorbance readings (duplicate)
DE Abs.
Absolute duplicate error
Ab s 1 , Ab s 2
Two absorbance readings, taken for each calibrator (duplicate readings)
All photometric calibrators are run in duplicate. If both the % error and the absorbance error are out of range, a Dup.E alarm is issued indicating a failed f ailed calibration. The following flowchart describes how a decision is made to flag a calibration for violating the duplication limit.
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Duplicate limit check (Dup.E)
Measure Abs1 and Abs2 for calibrator Std(N)
Compute
DE % and DE Abs.
Is DE Abs. <
Yes
ABS error limit?
No
Is DE % <
Yes
% error limit?
No Set Dup.E alarm
Fig Figure ure D-5
Dup.E limit flowchart
Continue
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7 Calculating data alarms Technical limit check (>Test)
Technical T echnical limit check (>T (>Test) est) If a result does not fall into the concentration range specified by the upper and lower Technical Limits on Utility > Application > Range, a data alarm >Test >Test or Test indicates results that exceed the upper limit. Range by by a concentration concentration conversion conversion coefficient coefficient (γ) as below. Equatio Equ ation n D-2
2 )1 ⋅ V ( S 1 )1 ⁄ [ V ( S 1 )1 + V (Dil ) 1 ] γ t 1 Range
t 2
Second Technical Limit entry on Utility > Application > Range
V ( S 1 )
Normal sample volume from a cup (or tube) to a cuvette when the primary sample type is diluted.
V (Dil )
Normal diluent volume for the primary sample type.
V ( S 2 )
Sample volume from cuvette to cuvette cuvette when the primary sample type is diluted.
V ( S 1 )1
Normal sample volume from a cup (or tube) to a cuvette when any sample type, with the exception of the primary sample type, is diluted.
V (Dil ) 1
Normal diluent volume for any sample type with the exception of the primary sample type.
V ( S 2 )1
Normal sample volume from cuvette to cuvette when any sample type, with the exception of the primary sample type, is diluted.
Primary is assigned to the sample type that is used during calibration. All other samp sample le types are corrected to the sample type used in calibration. When a sample is diluted, V ( S 1 )1 , V (Dil ) 1 and V ( S 2 )1 refer to the currently chosen sample type.
When a sample is not diluted, diluent volumes and S2 volumes (cuvette to cuvette) are zero. Therefore, γ in the equation above above simplifies to the following: 1 )1 γ = -V---(---S ---------V ( S 1 )
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Repeat limit check (>Rept)
Repeat limit check (>Rept) The Repeat Limit is checked with the final concentration (printed concen concentration). tration). Relationships between parameters on Utility Utility > Application > Range and check values of the Technical Technical Limit and Repeat Limit are shown below: below : Check item on Util iliit y > Appli lic catio ion n > Ran ge ge
Check valu e
Ch ec eck rang e
Technical Limit
[ t1 ] - [ t2 ]
Conc.1
[ t1 · γ ] - [ t2 · G ]
Repeat Limit
[ a1 ] - [ a2 ]
Conc.2
[ a1 ] - [ a2 ]
T Table able D-1
Parameters on Utility > Application > R ange and check values
Conc.1
Original concentration (measured) C 1
Conc.2
Final output concen concentration tration C 2 = ( 1 ⁄ γ ) ⋅ C 1
γ
Concentration conversion coefficient as calculated in the Technical Limit Checking section(a)
t1, t2
lower technical limit and upper technical limit
a1, a2
lower repeat limit and upper repeat limit
(a) See Technical limit check (>Test) on page page D-11 D-11..
When the result (Conc.1) is less than the lower Technical Technical Limit (t1), the t he result is flagged with a T >Test est data alarm. alar m. When the result (Conc.2) is less than the lower Repeat Limit (a1), the result is flagged with a Rept data alarm.
Reaction limit check (>React) In rate assays, correct data cannot obtained concentration activity value to is beyond the quantitative range. Forbe this reason, ifa the check is performedorwith reference a set upper or lower absorbance limit. For rate assays with ascending absorbances, the limit is an upper limit; for assays with descending absorbances, the limit is a lower l ower limit. The reaction reaction limit value is displayed on Utility > Application > Analyze.
Reaction Limit (upper) Reaction Limit (lower)
Fig Figure ure D-6
Reaction limits for ascending and descending rate assays
A data alarm (>React) is issued i ssued if only 3 or less measure points remain in the within the set absorbance limit. The alarm is not issued if there are 4 or more measure points within the absorbance limit.
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7 Calculating data alarms Reaction limit check (>React)
Data
Number of points
Photometric points
alarm
within Reaction Limit used for calculation
None
4
Reaction process
3 points within reaction limit
Photometric measurement range e c n a b r o s b A
Reaction Limit Time
>React
3
3 points within reaction limit
Photometric measurement range e c n a b r o s b A
Reaction Limit Time
>React
2 or less
First 2 points
Photometric measurement range e c n a b r o s b A
Reaction Limit Time T Table able D-2
Relationship between reaction limit check and photometric points
Automatic correction of Reaction Limit absorbance
The reaction limit check is performed with reference to the absorbance at the main wavelength. The analyzer automatically corrects the given reaction limit value by adding absorbance due to sample turbidity, etc.: Reaction limit absorbance after correction = Input reaction limit absorbance + (L1 - Lb) L1: Main wavelength absorbance of sample at photometric point 1 Lb: Main wavelength absorbance of reagent blank at photometric point 1 When L1 - Lb ≤ 0, the reaction limit absorbance is not corrected.
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Roche Diagnostics COBI C D · Version 1.0
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Quality control
8
E
Appl Applyin yingg QC QC rul rules es . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-3 E-3
cobas c 311 analyzer
8 Applying QC rules Table of contents
Applying QC rules
This chapter provides you with an overview of the application of quality control rules by the cobas c 311 analyzer analyzer.. The multi-rule Shewhart-type Shewhart-typ e method using the Westgard algorithm is described as well as possible alarms generated.
In this chapter
Chapter
8
Introduction ............................................................ ............................ .............................................................. ................................................... ..................... E-5 Rule 1: 1-2SD ....................................................................................... ......................................................... ..................................................... ....................... E-6 Rule 2: 11-2.5SD -2.5SD (Q2.5SD alarm) ..................................... .... .............................................................. .......................................... ............. E-6 Rule 3: 1-3SD (Q3SD alarm) .......................................... .......... ............................................................. ........................................... .............. E-7 Rule 4: 2-2SA (S2-2Sa alarm) ........................................................... ............................. ....................................................... ......................... E-8 Rule 5: R-4SD (R4SD alarm) ........................... .......................................................... .......................................................... ........................... E-9 Rule 6: 22-2SW -2SW (S2-2Sw alarm) ... .................................. ............................................................ .............................................. ................. E-10 Rule 7: 4-1SA (S4-1Sa alarm) ........................................................... ............................. ..................................................... ....................... E-11 Rule 8: 44-1SW -1SW (S4-1Sw alarm) ... .................................. ............................................................ .............................................. ................. E-12 Rule 9: 110XA 0XA (S1 (S10Xa 0Xa alarm) ................. ................................................ ........................................................... .................................... ........ E-13 Rule 10: 10 10XW XW (S10Xw (S10Xw alarm) .............................................................. ................................ ................................................ .................. E-14
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8 Applying QC rules Table of contents
cobas c 311 analyzer
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cobas c 311 analyzer
8 Applying QC rules Introduction
Introduction If selected in the software, the analyzer can utilize the Realtime QC to evaluate QC by a multi-rule Shewhart-type method using the W Westgard estgard algorithm. For each test, this algorithm algor ithm appli applies es a set of rules selected selected on QC > Individual Individual > Realtime Realtime QC > Rules Rules.. Any combination of rules may be specified. A pair of controls for each test being processed is compared against a known standard deviation (SD) and mean. If one or both of the controls fail a rule, the analyzer continues applying the testing criteria for all selected rules. When at least one rule violation is found, the appropriate data alarm for that rule is issued, and both the graph on o n the screen and the QC results on Workplace > Data Review are flagged. The QC alarm for the last rule violated is issued. The following is an explanation of each QC rule, using a display example where appropriate. All data alarms are listed in the Data alarms chapter of the Operator’s Manual. Control values Xn, Yn No
Inside control range
1-2SD Yes
No No
No 1-2.5S D Yes
1-3S D Yes
No
No 2-2SA Yes
R-4S D
No
No 2-2SW
Yes
Yes
4-1SA
4-1SW
Yes
Outside control range (error message) Fig Figure ure E-1
Application of Westgard rules
No
No
Yes
10XA Yes
10XW Yes
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Rule 1: 1-2SD
Rule 1: 1-2SD 1-2SD represents the control rule where one control result exceeds limits defined as the mean ± 2SD. Each control sample pair X and Y is compared against its respective expected mean and standard deviation. If both X and Y are within the mean ± 2SD, the QC results are accepted. No alarm is issued and no additional addit ional rules are checked for this control pair. If either X or Y results are outside the t he mean ± 2SD, the test fails, but no alarm is issued. The next selected rule is then applied and tested.
Rule 2: 1-2.5SD (Q2.5SD alarm) 1-2.5SD symbolizes the control rule violated when one control result exceeds the limit defined as the mean ± 2.5SD. If tighter QC restrictions are desired, this rule may be selected in place of Rule 2: 13SD. If both the 1-3SD and 1-2.5SD 1-2.5SD rules are selected in the QC > Individual > Realtime QC > Select Rules window and a set of controls fails both rules, the Q3SD alarm is issued. The deviation dev iation of a single sample is compared against 2.5 SD for each control. If either control X or Y is outside the mean ± 2.5 SD, the rule is violated. A Q2.5SD Q2.5 SD data alarm is issued issued for an indeterm indeterminate inate QC error error,, and a “ ” displays displays on the Yoden plot
. 1-2.5SD
Y 2.5SD
X
Are all X n and Y n in the cross-hatched area? Q2.5SD Fig Figure ure E-2
Q2.5SD alarm situation
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8 Applying QC rules Rule 3: 1-3SD (Q3SD alarm)
Rule 3: 1-3SD (Q3SD alarm) 1-3SD is the control rule violated when a single control X or Y result exceeds the limit defined as the mean ± 3SD. Each control sample X and Y is compared against its respective expected mean and standard deviation. If both X and Y are within the mean ± 3SD, the QC results are accepted. If either X or Y results are outside the mean ± 3SD, a Q3SD alarm is issued indicating an indeter indeterminat minatee QC error has occurre occurred. d. A “ ” displays displays on the screen screen in the appropriate part. 1-3SD
Y 3SD
X
Are all X n and Y n in the cross-hatched area? Q3SD Fig Figure ure E-3
Q3SD alarm situation
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Rule 4: 2-2SA (S2-2Sa alarm)
Rule 4: 2-2SA (S2-2Sa alarm) The results of one assay on each control are evaluated (a total of two control results are tested). The results of the most recent control pair of X and Y are compared against standard deviations. If both X and Y deviate outside ± 2SD and both are either above or below the mean, the rule is violated. A S2-2Sa data alarm is issued indicating a systematic QC erro errorr has occu occurre rred. d. A “ ” displa displays ys on the the Yode Yoden n plot. plot. The S2-2Sa alarm is issued when the two results (both (bot h X and Y in this t his case) are outside the ± 2SD limit, across control materials. This is a systematic violation.
2-2SD
Y 2SD
X
Are X n and Y n in the same cross-hatched area? S2-2Sa Fig Figure ure E-4
S2-2Sa alarm situation
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cobas c 311 analyzer
8 Applying QC rules Rule 5: R-4SD (R4SD alarm)
Rule 5: R-4SD (R4SD alarm) R-4SD is the control rule in which there is a range or a difference between the control materials that exceeds 4SD, as would be the case if the X control exceeded the -2SD limit and the Y control exceeded the +2SD limit. The run size specified when R-4SD R-4SD is selected on the QC > Individual > Realtime QC > Select Rules window determines the number of co consecutive nsecutive contr control ol X and Y samples tested. The maximum deviations of X minus the minimum deviations of Y, and the maximum deviations of Y minus the minimum deviations of X are computed. If either of these differences is greater than 4SD, the rule is violated. A R4SD data data alarm is issu issued ed for a random random QC QC error, error, and and a “ ” displays displays on the Yoden Yoden plot. R-4SD
Y 2SD
X
Are X n and Y n in the same cross-hatched area? (n is entered in the screen) R4SD Fig Figure ure E-5
R4SD alarm situation
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Rule 6: 2-2SW (S2-2Sw alarm)
Rule 6: 2-2SW (S2-2Sw alarm) The results of the two most recent assays of each control are evaluated. A total of four control results are tested. If either or both X and Y results deviate outside ± 2SD, the rule is violated. A S2-2Sw data alarm is issued indicating a systematic QC error has occurr occ urred. ed. A ” ” displa displays ys on the Yoden oden plot. plot. The S2-2Sw alarm is issued when two consecutive control results are outside of the 2SD limit, within a control material. This is a systematic violation. 2-2SD
Y
Y
-2SD
2SD 2SD
X
X
-2SD
Are X n and X n – 1 or
Y n and Y n – 1 in the same cross-hatched area? S2-2Sw Fig Figure ure E-6
S2-2Sw alarm situation
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cobas c 311 analyzer
8 Applying QC rules Rule 7: 4-1SA (S4-1Sa alarm)
Rule 7: 4-1SA (S4-1Sa alarm) 4-1SA is the control rule violated when four consecutive control results exceed the same limit, either mean + 1SD or mean - 1SD. The S4-1Sa alarm is issued when three control results are outside the ± 1SD limit and one control result is outside the ± 2SD limit across control materials. This is a systematic alarm. The results of two consecutive assays of each control are evaluated (total four samples tested). If insufficient data are available, the test is not performed. If all X and Y results exceed ± 1SD, and either the current X or Y value exceeds ± 2SD, and all are on the same side of the mean, then the rule is violated. A S4-1Sa data alarm is issued for a systematic syste matic QC al alarm, arm, and a ” ” displays displays on the the Yoden Yoden plot. 4-1SA
Y
1SD
X
Are X n , Y n , X n – 1 and
Y n – 1 in the same crosshatched area? S4-1Sa Fig Figure ure E-7
S4-1Sa alarm situation
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Rule 8: 4-1SW (S4-1Sw alarm)
Rule 8: 4-1SW (S4-1Sw alarm) The results of four consecutive assays of each control are evaluated (total of eight samples tested). If fewer than four samples are available, the test is not performed. If all X or Y results exceed one standard deviation, and fall on the same side si de of the mean, and the current X or Y value exceeds ± 2SD then the rule is violated. A S4-1Sw data alarm is is issued issued for a systema systematic tic QC aalarm, larm, and and a “ ” displays displays on the the Y Yoden oden plot. plot. The S4-1Sw alarm is issued when three consecutive control results are outside the ± 1SD limit and one control result is outside the ± 2SD limit wi within thin a control material. This is a systematic violation. 4-1SW
Y
-1SD
Y 1SD
1SD
X
X
-1SD
X n --33 or Are all X n to Y n to Y n --33 in the same cross-hatched area? S4-1Sw Fig Figure ure E-8
S4-1Sw alarm situation
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cobas c 311 analyzer
8 Applying QC rules Rule 9: 10XA (S10Xa alarm)
Rule 9: 10XA (S10Xa alarm) 10X is the control rule where there are 10 consecutive control observations (5 pairs) on the same side of the mean. The S10Xa alarm is issued when nine consecutive control results are on the same side of the mean and one control is outside the ± 2SD limit, across control materials. This is a systematic violation. The results of five consecutive assays of each control are evaluated (total 10 samples tested). If fewer than five samples are available for each control, the test is not performed. The signs of all sample deviations for both controls are compared with zero. If all are nonzero and have the same sign, and one of the current X and Y samples exceeds 2SD, 2SD, then the rule iiss violated. A S10Xa data processing alarm is issued issue d for a systematic systematic QC QC alarm, alarm, and a “ ” displays displays on on the Yoden plot. plot. 10XA
Y
X
Are all X n to X n – 4 and all Y n to Y n – 4 in the same cross-hatched area? S10Xa Fig Figure ure E-9
S10Xa alarm situation
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Rule 10: 10XW (S10Xw alarm)
Rule 10: 10XW (S10Xw alarm) The results of 10 consecutive assays of each control are evaluated (total 20 samples tested). If fewer than 10 samples are available for each control, the test is not performed. All sample deviations deviat ions are compared with zero. If all are nonzero and have the same sign, and the current sample (X or Y) exceeds ± 2SD, the rule is violated. A S10Xw S10X w data alarm is issued issued for for a systema systematic tic QC alarm, and a “ ” displays displays on on the Yoden plot. The S10Xw alarm is issued when nine consecutive control results are on the same side si de of the mean and one control result is outside out side the ± 2SD limit w within ithin a control material. This is a systematic violation. 10XW
Y
Y
X
X
Are all X n to X n – 9 and
Y n to Y n – 9 in the same cross-hatched area? S10Xw Fig Figure ure E-10 E-10
S10Xw alarm situation
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Index
F
cobas c 311 analyzer
Index
Index
A Absorbance limit, limit, rate assays, assays, D-12 Antigen readdition, D-5 Approvals, 2 Assay principles – ion selecti selective ve electrode, electrode, B-3 – photometric photometric:: See assay types. – serum index, index, B-47 Assay types, photometric – overview overview,, B-9 – 1 Poi Point, nt, B-24 – 2 Poi Point nt End, B-12 – 2 Point Point Rate, B-36 – Rate A, B-17 – Rate A with sample sample blank, blank, B-33 – summary summary,, B-44
B Bichromatic measurement, A-5 Blank calibration, C-17
D Data alarm calculation – absorbance absorbance limit, D-12 – calibrator calibrator duplicates, duplicates, D-9 – linearity linearity,, D-7 – repeat repeat limit, D-12 – sensitivity sensitivity limit, D-9 – substrate substrate depletion, depletion, D-12 – technical technical limit, D-11 Document information, 2 Duplicate limit, D-9
E Edition notice, 2 Electromotive force (EMF), B-5 EMF (electromotive (electromotive force), B-5 Endpoint assays – 1 Point, Point, B-24 – 2 Point Point End, B-27
F C C. Value, Value, ISE calibration, C-7 Calibration methods, photometric – 2 Point Point calibration, calibration, C-18 – Blank calibration, calibration, C-17 – Full calibration, calibration, C-18 – Span calibration, calibration, C-17 Calibration types, photometric, C-15 Calibration update types, photometric, C-19 Cell Blank Measurements Measurements report, B-21 Concentration calculation – 1 Point assay, assay, B-26 – 2 Point Point End assay, assay, B-28 – 2 Point Point Rate assay, assay, B-36 – Rate A assay assay,, B-31 Contact addresses, 3 Copyrights, 2
Full calibration, C-18
H Hemolysis index, index, calculation, calculation, B-50
I Icterus index, ca calculation, lculation, B-50 Instrument approvals, 2 Intended use, 2 Internal standard calculation, calculation, C-6 ISE calibration – internal internal standard, standard, C-6 – reference electrode, C-8 – slope calcu calculation lation,, C-6 ISE reference reference electrode, C-8 ISE, calculating concentrations, B-5
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Index
K K factor – calculation calculation,, C-19 – definition, definition, C C-15 -15
L Line Graph calibration, C-33 Linear 2 point calibration, C-22 Linearity verification, D-7 Lipemia index, index, calculation, calculation, B-50
N Non-linear calibration – Line Graph, Graph, C-33 – RCM, RCM, C-25 C-25 – RCM2T1, RCM2T1, CC-27 27 – RCM2T2, RCM2T2, CC-29 29 – Spline, Spline, C-31 C-31
O One-point calibration – ISE, ISE, C-7 C-7 – photometric photometric test, C-17 Others tab, Utility Utility > Application screen, screen, B-23
P Photometer – general characteristics, A-5 – light path, A-5 Photometric assays – assay assay types, B-9 Photometric calibration – overview overview,, C-14 – calibration calibration methods, methods, C-16 – calibration calibration types, C-15 – calibration calibration update types, C-19 Prozone check – antigen antigen readdition, readdition, B-39 – check value calculation, B-41 – reaction reaction rate metho method, d, B-42 Prozone effect, definition, D-5
cobas c 311 analyzer
Q QC rules – rule 1: 1-2SD, 1-2SD, E-6 – rule 2: 1-2.5SD 1-2.5SD,, E-6 – rule 3: 1-3SD, 1-3SD, E-7 – rule 4: 2-2SA, 2-2SA, E-8 – rule 5: R-4SD, R-4SD, E-9 – rule 6: 2-2SW, 2-2SW, E-10 – rule 7: 4-1SA, 4-1SA, E-11 – rule 9: 10XA, 10XA, E-13 – rule10: 10XAW 10XAW, E-14
R Rate assays – 2 Point Point Rate, B-36 – Rate A with sample sample blank, blank, B-33 RCM calibration, C-25 RCM2T1 calibration, C-27 RCM2T2 calibration, C-29 Reaction limit, D-12 Reaction rate method, method, D-6 Realtime QC, E-5 Repeat limit, D-12 Revision History, 2
S Sensitivity limit, D-9 Serum index – calculation calculation,, B-50 – data alarms, alarms, B-51 – definition, definition, BB-49 49 – principles, principles, B-47 B-47 Shewhart multi-rule multi-rule method, E-5 Span calibration, C-17 Spline calibration, C-31 Substrate depletion, D-12
T Technical limit, D-11 Trademarks, 2 Two-point calibration, C-18
W Weighting, photometric calibration, C-21 Westgard rules, E-5 Working Information window, B B-22 -22
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