Calibration/Verification and Determination of Cholesterol in human serum and plasma using cobas

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Biomonitoring of Great Lakes Populations Program II

Calibration/Verification and Determination of Cholesterol in human serum and plasma using cobas

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Wadsworth Center
Clinical Chemistry
Calibration Verification
Roche cobas c501

SOP CLC-09.0
Effective Date: December 1, 2010
Page 1 of 5

1. Purpose
This procedure outlines the protocol to be used to perform calibration verification
of test methods employed on the Roche cobas c501.
2. Summary and explanation
Calibration verification is performed using calibration materials appropriate for the
methodology in use and also can be used to verify the reportable range of a
method if materials span the reportable range. At a minimum, three samples
containing analyte concentrations of a zero or minimal value, a midpoint, and a
maximum value should be used for each analyte.
Calibration verification must be performed in accordance with the manufacturer’s
calibration verification instructions if provided or in accordance with the criteria
established by the laboratory at least every six months or whenever any of the
following occur:


Major preventative maintenance or replacement of critical parts is performed
which may influence test performance.



A complete change of reagents affects assay performance (i.e. reportable
ranges and/or control values are adversely affected by reagent lot changes).



Controls reflect an unusual shift or trend or are outside the acceptable limits
and other means of correcting the problem have not been effective.

3. Special Safety Notes/Warnings/Precautions
Follow all procedures for handling and disposal of potentially biohazardous
materials described in the Wadsworth Center Safety & Security Policy &
Procedures Manual. Disposable gloves and protective clothing (lab coat) must
be worn at all times when operating the Roche cobas c501 clinical analyzer.
Gloves should be changed as necessary but always if torn or soiled. Wash
hands thoroughly after removing gloves. A splash barrier capable of protecting
the face should be used whenever performing manipulations that might produce
splashes of potentially biohazardous materials. Note: The following are
considered Aclean areas@: instrument and cobasLink keyboard, monitor, printer,
and all bench areas not covered by blue absorbent pads. Contact with these
areas while wearing disposable gloves should be avoided in order to prevent
contamination.
4. Equipment/Materials/Reagents

Roche cobas c501 analyzer
Roche cobas c501 reagents
Calibrator(s) (see application information for appropriate material)
Controls (see application information for appropriate material)
Calibration verification materials (see CLC-09.0 Appendix A,B,C)
NYS DOH Wadsworth Center Controlled Document

Wadsworth Center
Clinical Chemistry
Calibration Verification
Roche cobas c501

SOP CLC-09.0
Effective Date: December 1, 2010
Page 2 of 5

Procedure
4.1

Perform all required maintenance procedures on the Roche cobas c501.

4.2

Review on-board reagents to verify sufficient quantity and stability. Load
fresh reagents as necessary.

4.3

Perform required calibration (see CLC-SOP 05.0) and quality control (see
CLC-SOP 06.0). Review calibration and quality control results. If
acceptable, proceed to calibration verification.

4.4

Follow all calibration verification material instructions for storage, thawing,
reconstitution and stability. Each level of material must be run at least in
duplicate.

4.5

Program calibration verification materials/test selections into the Roche
cobas c501.

4.6

After all tests/samples have been entered into the cobas c501 test
selection screen, fill labeled sample cups with the appropriate calibration
verification material and load them into the corresponding rack/sample
position. Check that there are no bubbles or foam visible on the liquid
surface of each sample.

4.7

Click START button; then click large START button to begin analysis.

4.8

When testing is complete, review results to determine whether off-line
dilutions of calibration verification materials should be made to closer
approximate lower and/or upper reportable range limits. Print copy of
results, reagent listing, and requisition list for filing.

4.9

Enter calibration verification data into the Microgenics/CASCO on-line
data reduction software (see web address below). Alternate software
products may be used if they provide similar functionality.
http://www.cascodocservices.com/admin/login.aspx?ReturnUrl=%2fDefau
lt.aspx).
4.9.1

Print results and review as outlined below under “Interpretation of
Results”.

4.9.2

Analyst must sign calibration verification document and forward to
supervisor and/or director for review.

4.9.3

After results are reviewed, accepted, and signed by supervisor
(and/or director), file evaluation printout in Calibration Verification
binder. Be certain to check off and indicate date of calibration
verification on binder summary sheet.

NYS DOH Wadsworth Center Controlled Document

Wadsworth Center
Clinical Chemistry
Calibration Verification
Roche cobas c501
5.

SOP CLC-09.0
Effective Date: December 1, 2010
Page 3 of 5

Interpretation of Results
5.1

Agreement of measured results with expected values should be ≤ 10% for
all levels, with exceptions at the extreme lower limits where a small bias
may result in a large % difference due to the very low concentration of the
analyte. Thus, the 10% criteria cannot always be used for the minimum
calibration verification point, especially when the predicted value is very
low. In such cases, professional judgment and consultation with
supervisor and/or director must be used to determine acceptability.

5.2

Confirm that manufacturer’s reportable range limits (refer to the
manufacturer’s test method application sheet) have been verified.

5.3

In the event calibration verification materials do not span the reportable
range OR if results for the upper and/or lower point(s) fall out of range,
consult supervisor and/or director. In such cases, the director and/or
supervisor may determine that decreasing the reportable range to the
limits verified is acceptable.
5.3.1

Should decreasing manufacturer’s claimed reportable range be
necessary and approved by supervisor and/or director, make
certain to note adjusted ranges on:




5.4

Roche method application sheet (obtain director’s signature)
Roche c501 method parameters (Utilities/Application screen).
Be certain to note change in Parameter Update Log book.
CLC-02.0 Appendix A, B, or C (as appropriate) Clinical
Chemistry, Urine Chemistry, TSM Verified Ranges.

Should calibration verification results fall outside expected tolerances,
perform corrective action as required and repeat calibration verification
study for the analyte in question. Consult supervisor and/or director if
repeat testing after corrective action(s) fails to result in acceptable
performance.

6. References
cobas® 6000 analyzer series Operator’s Manual. Roche Diagnostics GmbH. ©
2001-2009. Version 4.0
New York State Department of Health, Clinical Laboratory Standards of Practice.
January 2008.
Wadsworth Center Safety & Security Policy & Procedures Manual

NYS DOH Wadsworth Center Controlled Document

Wadsworth Center
Clinical Chemistry
Calibration Verification
Roche cobas c501

SOP CLC-09.0
Effective Date: December 1, 2010
Page 4 of 5

7. Appendix
CLC-09.0 Appendix A Clinical Chemistry Calibration Verification Materials
CLC-09.0 Appendix B Urine Chemistry Calibration Verification Materials
CLC-09.0 Appendix C TSM Calibration Verification Materials

NYS DOH Wadsworth Center Controlled Document

Wadsworth Center
Clinical Chemistry

SOP CLC-09.0

Calibration Verification
Roche cobas c501

Effective Date: December 1, 2010
Page 5 of 5

SOP Annual Review

Director or Designee

Signature

NYS DOH Wadsworth Center Controlled Document

Date

Wadsworth Center
Clinical Chemistry
Calibration
Roche cobas c501

SOP CLC-05.0

Effective Date: December 1, 2010
Page 1 of 5

1. Purpose
This procedure describes calibration requirements for the Roche cobas c501
clinical analyzer and details the mechanism used to determine when calibration
must be performed, the method used to perform calibration, and the criteria
employed to accept or reject a calibration.
2. Summary and explanation
The Roche cobas c501 is a fully automated system for photometric analysis.
Calibration is the process that establishes a relationship between measurement
values (such as absorbance values) and corresponding results (concentration of
an analyte). The relationship between measurement values and results is subject
to various environmental and reagent conditions and may drift over the course of
time. It is thus necessary to repeat calibrations regularly. To keep the resulting
calibration management simple and efficient, the Roche cobas c501 system
automatically recommends calibrations.
This procedure provides an outline of the steps that must be performed in order
to perform calibrations on the Roche cobas c501. The procedures described in
this document have been obtained from the Roche cobas 6000 Operator’s
Manual and can be reviewed in greater detail by consulting appropriate sections
of that manual and/or the cobas 6000 analyzer series Quick Reference Card for
calibrations (CLC-10.0 Appendix A).
3. Equipment/Materials/Reagents
Roche cobas c501 clinical analyzer
Reagent cassettes for applicable tests
Calibrator(s) as required (see CLC-05.0 Appendix A)
Quality control materials as required (see SOP CLC-06.0)
4. Special Safety Notes/Warnings/Precautions
Follow all procedures for handling and disposal of potentially biohazardous
materials described in the Wadsworth Center Safety & Security Policy &
Procedures Manual. Disposable gloves and protective clothing (lab coat) must
be worn at all times when operating the Roche cobas c501 clinical analyzer.
Gloves should be changed as necessary but always if torn or soiled. Wash
hands thoroughly after removing gloves. A splash barrier capable of protecting
the face should be used whenever performing manipulations that might produce
splashes of potentially biohazardous materials. Note: The following are
considered Aclean areas@: instrument and cobasLink keyboard, monitor, printer,
and all bench areas not covered by blue absorbent pads. Contact with these
areas while wearing disposable gloves should be avoided in order to prevent
contamination.
NYS DOH Wadsworth Center Controlled Document

Wadsworth Center
Clinical Chemistry
Calibration
Roche cobas c501

SOP CLC-05.0

Effective Date: December 1, 2010
Page 2 of 5

5. Procedure
The number and type of calibrators varies and is dependent upon the test to be
calibrated. Calibration data are automatically downloaded via the cobasLink and
then downloaded by the operator into the cobas c501 control unit. This data may
also be entered manually.
Calibrators do not contain sample ID bar codes. However, each calibrator has
been assigned specific rack and position numbers. Each calibrator rack has
been pre-labeled with the calibrator names to aid in correct calibrator placement.
5.1

ISE Calibration (Na, K, and Cl)
ISE calibration is required every 24 hours, after ISE
cleaning/maintenance, after changing the ISE reagent solutions or
cartridges, and as required following quality control troubleshooting
protocols.
5.1.1

From the System Overview screen click CALIBRATION

5.1.2

Click the STATUS tab to display the CALIBRATION STATUS
SCREEN.

5.1.3

Select ISE-A-IS1 and ISE-B-IS1. The selected line is highlighted
in blue.

5.1.4

Select the appropriate button in the METHOD area for a FULL
calibration. The selected option appears in the METHOD column
highlighted in green, the CAUSE column indicates MANUAL, and
the SAVE button turns yellow.

5.1.5

Click SAVE to save the changes.

5.1.6

Click the PRINT button and then select CALIBRATOR LOAD
LIST, then click PRINT to print a detailed list of the calibrators and
their corresponding rack positions to assist in loading those on the
analyzer.

5.1.7

Calibrators for sodium, potassium, and chloride are supplied in as
ready-to-use liquids. Consult Appendix APP CLC-00.0) for a list
of tests and their associated calibrators. Current calibrator value
sheets/inserts are filed in the “Calibrator Inserts” binder.

5.1.8

Fill labeled sample cups with the calibrators and place those into
the appropriate rack/rack positions. Check that there are no
bubbles or foam visible on the liquid surface.

NYS DOH Wadsworth Center Controlled Document

Wadsworth Center
Clinical Chemistry
Calibration
Roche cobas c501
5.1.9

5.2

SOP CLC-05.0

Effective Date: December 1, 2010
Page 3 of 5

Load the calibrator rack and onto the Roche cobas c501. Select
START, and then click the large START button to begin the
calibration.

Non-ISE Calibrations
Calibration is generally required after reagent lot change, at specific testspecific time intervals after initial lot calibration, and as required following
quality control troubleshooting protocols. For specific calibration
requirements, refer to the individual analyte application sheets.
5.2.1

From the main screen select CALIBRATION.

5.2.2

Click STATUS tab to display the CALIBRATION STATUS
SCREEN.

5.2.3

Select the reagent and test to be calibrated from the list. The
selected line is highlighted in blue.

5.2.4

Select the appropriate button in the METHOD area for a 2-point,
full, or blank calibration. The selected option appears in the
METHOD column highlighted in green, the CAUSE column
indicates MANUAL, and the SAVE button turns yellow.

5.2.5

Click SAVE to save the changes.

5.2.6

Click the PRINT button and then select CALIBRATOR LOAD
LIST, then click PRINT to print a detailed list of the calibrators and
their corresponding rack positions. This list will assist the operator
in loading the appropriate calibrators on the Roche c501.

5.2.7

Prepare calibrator(s) as required for the test to be calibrated.
Calibrators are supplied in either lyophilized form or as ready-touse liquids. Consult Appendix CLC-05.0 Appendix A) for a list of
tests and their associated calibrators. Current calibrator value
sheets/inserts are filed in the “Calibrator Inserts” binder.

5.2.8

Fill labeled sample cups with the appropriate calibrators and place
them into the corresponding rack(s). Check that there are no
bubbles or foam visible on the liquid surface.

5.2.9

Once loaded on the instrument, click START, and then click the
large START button to begin the calibration.

NYS DOH Wadsworth Center Controlled Document

Wadsworth Center
Clinical Chemistry
Calibration
Roche cobas c501

SOP CLC-05.0

Effective Date: December 1, 2010
Page 4 of 5

6. Interpretation of Results
6.1

Calibration results will automatically be assessed and printed by the
analyzer. Make certain to review the results, checking for calibration
alarms and/or unusual results, and date/initial calibration printout. In
general, quality control samples are analyzed immediately after a
calibration is performed, and should be used as an additional check of the
validity of the calibration performed.

6.2

Should an unacceptable calibration occur, proceed as follows before
commencing patient testing:
6.2.1

Check that appropriate calibrator(s) were loaded in the correct
rack/position.

6.2.2

Check that calibrator(s) dating is within specified expiration date
and that reconstitution (if required) was performed correctly.

6.2.3

Check reagent expiration and on-board stability.

6.2.4

Check that all maintenance is up-to-date and acceptable.

6.2.5

Repeat calibration and quality control samples.

6.2.6

If calibration continues to fail, install fresh reagent and/or reagent
cassette and repeat calibration and quality control.

6.2.7

Consult supervisor and/or Roche Technical Support if the above
remedies fail to correct calibration problems.

7. References
cobas® 6000 analyzer series Operator’s Manual. Roche Diagnostics GmbH. ©
2001-2009. Version 4.0
Wadsworth Center Safety & Security Policy & Procedures Manual
8. Appendix
SOP CLC-05.0A Roche cobas c501 Calibrator/Test list

NYS DOH Wadsworth Center Controlled Document

Wadsworth Center
Clinical Chemistry
Calibration
Roche cobas c501

SOP CLC-05.0

Effective Date: December 1, 2010
Page 5 of 5

SOP Annual Review

Director or Designee

Signature

NYS DOH Wadsworth Center Controlled Document

Date

Wadsworth Center
Clinical Chemistry

SOP CLC-11.0

Method Validation

Effective Date: December 1, 2010

Roche cobas c501

Page 1 of 3

1. Purpose
This procedure provides a description of the protocol to be followed for validation
of any new method and/or instrumentation.
2. Summary and explanation
Method validation is performed in order to verify the manufacturer’s claims for
precision, accuracy, and reportable range of all new methods and/or
instrumentation. Validation results must be reviewed and accepted by the
laboratory director before patient testing can commence. Records from
validation studies must be retained while a procedure is in use and for two years
after the procedure has been discontinued.
3. Equipment/Materials/Reagents
Roche cobas c501 clinical analyzer
Reagent cassettes for applicable tests
Calibrator(s)
Quality control materials
Patient and/or proficiency test specimens
4. Special Safety Notes/Warnings/Precautions
Follow all procedures for handling and disposal of potentially biohazardous
materials described in the Wadsworth Center Safety & Security Policy &
Procedures Manual. Disposable gloves and protective clothing (lab coat) must
be worn at all times when operating the Roche cobas c501 clinical analyzer.
Gloves should be changed as necessary but always if torn or soiled. Wash
hands thoroughly after removing gloves. A splash barrier capable of protecting
the face should be used whenever performing manipulations that might produce
splashes of potentially biohazardous materials. Note: The following are
considered Aclean areas@: instrument and cobasLink keyboard, monitor, printer,
and all bench areas not covered by blue absorbent pads. Contact with these
areas while wearing disposable gloves should be avoided in order to prevent
contamination.
5. Procedure
5.1

Assessment of precision
5.1.1

Refer to SOP CLC-12.0 Precision Assessment-Within Run.
Values for standard deviation/coefficient of variation should fall
within manufacturer=s specifications.

NYS DOH Wadsworth Center Controlled Document

5.2

5.3

Wadsworth Center
Clinical Chemistry

SOP CLC-11.0

Method Validation

Effective Date: December 1, 2010

Roche cobas c501

Page 2 of 3

Assessment of accuracy
5.2.1

Analyze assayed quality control materials provided by the
manufacturer. If results do not fall within allowable limits, consult
Calibration and Quality Control procedures (SOP CLC-05.0 and
SOP CLC-06.0) for troubleshooting advice.

5.2.2

Analyze proficiency test specimens (eg. NYS, CAP) and compare
measured values with target values and allowable ranges for
similar reagent/instrument systems. Results must fall within
acceptable limits.

Confirmation of reportable range/calibration verification
5.3.1

5.4

Refer to SOP CLC-09.0 Calibration Verification. Results should
confirm manufacturer=s reportable range claims for all methods
tested. If calibration verification materials do not span the
manufacturer’s stated reportable ranges, adjust in-house reporting
limits accordingly.

Director review and sign-off
5.4.1

Assemble all precision, accuracy, and reportable range data and
summaries for director review.

5.4.2

Obtain a printed copy of the Roche cobas 6000 application sheet
for the test undergoing validation and include with validation data
for director review and signature.

5.4.3

After all documents/data have been reviewed and signed by the
director, file validation summaries in the cobas c501 installation
binder. File signed Roche cobas application sheet in the Method
Manual (SOP CLC-02.0)

6. References
Wadsworth Center Safety & Security Policy & Procedures Manual

NYS DOH Wadsworth Center Controlled Document

Wadsworth Center
Clinical Chemistry

SOP CLC-11.0

Method Validation

Effective Date: December 1, 2010

Roche cobas c501

Page 3 of 3
SOP Annual Review

Director or Designee

Signature

NYS DOH Wadsworth Center Controlled Document

Date

Application Sheet
Laboratory Name
Test Name: Cholesterol Gen.2
y Indicates cobas c systems on which reagents can be used
Order information
Cholesterol Gen.2
400 tests
Calibrator f.a.s. (12 x 3 mL)
Calibrator f.a.s. (12 x 3 mL,
for USA)
Precinorm U plus (10 x 3 mL)
Precinorm U plus (10 x 3 mL,
for USA)
Precipath U plus (10 x 3 mL)
Precipath U plus (10 x 3 mL,
for USA)
Precinorm U (20 x 5 mL)
Precipath U (20 x 5 mL)
Precinorm L (4 x 3 mL)
Precipath L (4 x 3 mL)
Diluent NaCl 9 % (50 mL)

Cat. No. 03039773 190
Cat. No. 10759350 190
Cat. No. 10759350 360

System-ID 07 6726 3
Code 401
Code 401

Cat. No. 12149435 122
Cat. No. 12149435 160

Code 300
Code 300

Cat. No. 12149443 122
Cat. No. 12149443 160

Code 301
Code 301

Cat. No. 10171743 122
Cat. No. 10171778 122
Cat. No. 10781827 122
Cat. No. 11285874 122
Cat. No. 04489357 190

Code 300
Code 301
Code 304
Code 305
System-ID 07 6869 3

Effective date
Effective date for this procedure: ____________________________

Author
Source documentation compiled by Roche Diagnostics
Revised by: ___________________________________________

Schedule for review
Last date revised: __________________________________________
Date Reviewed: _____________ Approved: _____________________
Date Reviewed: _____________ Approved: _____________________
Date Reviewed: _____________ Approved: _____________________
Date Reviewed: _____________ Approved: _____________________

System information
For cobas c 311/501 analyzers:
CHO2I: ACN 798: ID/MS Standardization
CHO2A: ACN 433: Abell/Kendall Standardization
For cobas c 502 analyzer:
CHO2I: ACN 8798: ID/MS Standardization
CHO2A: ACN 8433: Abell/Kendall Standardization
1 of 9 pages

Roche/Hitachi
cobas c systems
cobas c
cobas c
311
501/502
y
y

Laboratory Name
Test Name: Cholesterol Gen.2

Intended use
In vitro test for the quantitative determination of cholesterol in human serum and plasma on Roche/Hitachi
cobas c systems.

Summary
Cholesterol is a steroid with a secondary hydroxyl group in the C3 position. It is synthesized in many types
of tissue, but particularly in the liver and intestinal wall. Approximately three quarters of cholesterol is newly
synthesized and a quarter originates from dietary intake. Cholesterol assays are used for screening for
atherosclerotic risk and in the diagnosis and treatment of disorders involving elevated cholesterol levels as
well as lipid and lipoprotein metabolic disorders.
Cholesterol analysis was first reported by Liebermann in 1885 followed by Burchard in 1889. In the
Liebermann-Burchard reaction, cholesterol forms a blue-green dye from polymeric unsaturated
carbohydrates in an acetic acid/acetic anhydride/concentrated sulfuric acid medium. The Abell and Kendall
method is specific for cholesterol, but is technically complex and requires the use of corrosive reagents. In
1974, Roeschlau and Allain described the first fully enzymatic method. This method is based on the
determination of Δ4-cholestenone after enzymatic cleavage of the cholesterol ester by cholesterol esterase,
conversion of cholesterol by cholesterol oxidase, and subsequent measurement by the Trinder reaction of the
hydrogen peroxide formed. Optimization of ester cleavage (> 99.5 %) allows standardization using primary
and secondary standards and a direct comparison with the CDC and NIST reference methods.1,2,3,4,5,6,7,8,9
Nonfasting sample results may be slightly lower than fasting results.10,11,12
The Roche cholesterol assay meets the 1992 National Institutes of Health (NIH) goal of less than or equal to
3 % for both precision and bias.12
The assay is optionally standardized against Abell/Kendall and isotope dilution/mass spectrometry. The
performance claims and data presented here are independent of the standardization.

Test principle
Enzymatic, colorimetric method.
Cholesterol esters are cleaved by the action of cholesterol esterase to yield free cholesterol and fatty acids.
Cholesterol oxidase then catalyzes the oxidation of cholesterol to cholest-4-en-3-one and hydrogen peroxide.
In the presence of peroxidase, the hydrogen peroxide formed effects the oxidative coupling of phenol and 4aminophenazone to form a red quinone-imine dye.
CE

Cholesterol esters + H2O

cholesterol + RCOOH
CHOD

Cholesterol + O2

cholest-4-en-3-one + H2O2
POD

2 H2O2 + 4-AAP + phenol

quinone-imine dye + 4 H2O

The color intensity of the dye formed is directly proportional to the cholesterol concentration. It is
determined by measuring the increase in absorbance.

2 of 9 pages

Laboratory Name
Test Name: Cholesterol Gen.2

Reagents – working solutions
R1 PIPES buffer: 225 mmol/L, pH 6.8; Mg2+: 10 mmol/L; sodium cholate: 0.6 mmol/L;
4-aminophenazone: ≥ 0.45 mmol/L; phenol: ≥ 12.6 mmol/L; fatty alcohol polyglycol ether: 3 %;
cholesterol esterase (Pseudomonas spec.): ≥ 25 µkat/L (≥ 1.5 U/mL); cholesterol oxidase (E. coli):
≥ 7.5 µkat/L (≥ 0.45 U/mL); peroxidase (horseradish): ≥ 12.5 µkat/L (≥ 0.75 U/mL); stabilizers;
preservative

Reagent handling
Ready for use.

Storage and stability
CHOL2
Shelf life at 2-8 °C:
On-board in use and refrigerated on the analyzer:

See expiration date on cobas c pack label.
4 weeks

Diluent NaCl 9 %
Shelf life at 2-8 °C:
On-board in use and refrigerated on the analyzer:

See expiration date on cobas c pack label.
12 weeks

Specimen collection and preparation
For specimen collection and preparation, only use suitable tubes or collection containers.
Only the specimens listed below were tested and found acceptable.
Serum.
Plasma: Li-heparin and K2-EDTA plasma
Do not use citrate, oxalate or fluoride.13
Fasting and nonfasting samples can be used.11
The sample types listed were tested with a selection of sample collection tubes that were commercially
available at the time of testing, i.e. not all available tubes of all manufacturers were tested. Sample collection
systems from various manufacturers may contain differing materials which could affect the test results in
some cases. When processing samples in primary tubes (sample collection systems), follow the instructions
of the tube manufacturer.
Centrifuge samples containing precipitates before performing the assay.
Stability:14,15
7 days at 15-25 °C
7 days at 2-8 °C
3 months at (-15)-(-25) °C

3 of 9 pages

Laboratory Name
Test Name: Cholesterol Gen.2

Materials provided
See “Reagents – working solutions” section for reagents.

Materials required (but not provided)
See “Order information” section.
General laboratory equipment
Other suitable control material can be used in addition.
_______________________________________________________________________________________
_______________________________________________________________________________________

Assay
For optimum performance of the assay follow the directions given in this document for the analyzer
concerned. Refer to the appropriate operator's manual for analyzer-specific assay instructions.
The performance of applications not validated by Roche is not warranted and must be defined by the user.

Application for serum and plasma
cobas c 311 test definition
Assay type
Reaction time / Assay points
Wavelength (sub/main)
Reaction direction
Units
Reagent pipetting
R1
Sample volumes
Normal
Decreased
Increased
cobas c 501/502 test definition
Assay type
Reaction time / Assay points
Wavelength (sub/main)
Reaction direction
Units
Reagent pipetting
R1
Sample volumes
Normal
Decreased
Increased

1 Point
10 / 57
700/505 nm
Increase
mmol/L (mg/dL, g/L)
47 µL
Sample
2 µL
2 µL
4 µL

Diluent (H2O)
93 µL
Sample dilution
Sample
Diluent (NaCl)
–
–
15 µL
135 µL
–
–

1 Point
10 / 70
700/505 nm
Increase
mmol/L (mg/dL, g/L)
47 µL
Sample
2 µL
2 µL
4 µL

4 of 9 pages

Diluent (H2O)
93 µL
Sample dilution
Sample
Diluent (NaCl)
–
–
15 µL
135 µL
–
–

Laboratory Name
Test Name: Cholesterol Gen.2

Calibration
Calibrators
Calibration mode
Calibration frequency

S1: H2O
S2: C.f.a.s.
Linear
2-point calibration
• after reagent lot change
• and as required following quality control procedures

Traceability: This method has been standardized according to Abell/Kendall12 and also by isotope
dilution/mass spectrometry.16

Quality Control
For quality control, use control materials as listed in the “Order information” section.
Other suitable control material can be used in addition.
The control intervals and limits should be adapted to each laboratory’s individual requirements. Values
obtained should fall within the defined limits. Each laboratory should establish corrective measures to be
taken if values fall outside the limits.
Follow the applicable government regulations and local guidelines for quality control.
If controls do not recover within the specified limits, take the following corrective action:
_______________________________________________________________________________________
_______________________________________________________________________________________

Calculation
Roche/Hitachi cobas c systems automatically calculate the analyte concentration of each sample.
Conversion factors:
mmol/L x 38.66 = mg/dL
mmol/L x 0.3866 = g/L
mg/dL x 0.0259 = mmol/L

Limitations – interference17
Criterion: Recovery within ± 10 % of initial values at a cholesterol concentration of 5.2 mmol/L
(200 mg/dL).
Icterus: No significant interference up to an I index of 16 for conjugated bilirubin and 14 for unconjugated
bilirubin (approximate conjugated bilirubin concentration 274 µmol/L (16 mg/dL) and approximate
unconjugated bilirubin concentration 239 µmol/L (14 mg/dL)).
Hemolysis: No significant interference up to an H index of 700 (approximate hemoglobin concentration:
435 µmol/L (700 mg/dL)).
Lipemia (Intralipid): No significant interference up to an L index of 2000. There is poor correlation between
the L index (corresponds to turbidity) and triglycerides concentration.
Drugs: No interference was found at therapeutic concentrations using common drug panels.18,19
In very rare cases, gammopathy, in particular type IgM (Waldenström's macroglobulinemia), may cause
unreliable results.
For diagnostic purposes, the results should always be assessed in conjunction with the patient's medical
history, clinical examination and other findings.

5 of 9 pages

Laboratory Name
Test Name: Cholesterol Gen.2
ACTION REQUIRED
Special Wash Programming: The use of special wash steps is mandatory when certain test combinations are
run together on Roche/Hitachi cobas c systems. The latest version of the Carry over evasion list can be
found with the NaOHD/SMS/Multiclean/SCCS or the NaOHD/SMS/SmpCln1 + 2/SCCS Method Sheets.
For further instructions refer to the operator manual.
cobas c 502 analyzer: All special wash programming necessary for avoiding carry over is available via the
cobas link, manual input is not required.
Where required, special wash/carry over evasion programming must be implemented prior to
reporting results with this test.

Limits and ranges
Measuring range
0.1-20.7 mmol/L (3.86-800 mg/dL)
Determine samples having higher concentrations via the rerun function. Dilution of samples via the rerun
function is a 1:10 dilution. Results from samples diluted by the rerun function are automatically multiplied
by a factor of 10.
Lower limits of measurement
Lower detection limit of the test
0.1 mmol/L (3.86 mg/dL)
The lower detection limit represents the lowest measurable analyte level that can be distinguished from zero.
It is calculated as the value lying three standard deviations above that of the lowest standard (standard
1 + 3 SD, repeatability, n = 21).

Expected values
Clinical interpretation according to the recommendations of the European Atherosclerosis Society:20
Cholesterol
Triglycerides
Cholesterol
Cholesterol
Triglycerides

mmol/L
< 5.2
< 2.3

mg/dL
(< 200)
(< 200)

Lipid metabolic disorder

5.2-7.8

(200-300)

Yes, if HDL-cholesterol < 0.9 mmol/L
(< 35 mg/dL)

> 7.8
> 2.3

(> 300)
(> 200)

Yes

Recommendations of the NCEP Adult Treatment Panel for the following risk-cutoff thresholds for the US
American population:21
Desirable cholesterol level
< 5.2 mmol/L
(< 200 mg/dL)
Borderline high cholesterol
5.2-6.2 mmol/L
(200-240 mg/dL)
High cholesterol
≥ 6.2 mmol/L
(≥ 240 mg/dL)
Each laboratory should investigate the transferability of the expected values to its own patient population and
if necessary determine its own reference ranges.

Specific performance data
Representative performance data on the analyzers are given below. Results obtained in individual
laboratories may differ.

6 of 9 pages

Laboratory Name
Test Name: Cholesterol Gen.2

Precision
Precision was determined using human samples and controls in an internal protocol. Repeatability* (n = 21),
intermediate precision** (3 aliquots per run, 1 run per day, 21 days). The following results were obtained:
Repeatability *
Mean
SD
CV
mmol/L (mg/dL)
mmol/L (mg/dL)
%
Precinorm U
2.29 (88.5)
0.02 (0.8)
1.1
Precipath U
4.74 (183)
0.04 (2)
0.9
Human serum 1
2.85 (110)
0.03 (1)
1.1
Human serum 2
7.39 (286)
0.05 (2)
0.7
Intermediate precision **
Precinorm U
Precipath U
Human serum 3
Human serum 4

Mean
mmol/L (mg/dL)
2.31 (89.3)
4.85 (188)
1.97 (76.2)
7.13 (276)

SD
mmol/L (mg/dL)
0.04 (1.6)
0.08 (3)
0.03 (1.2)
0.10 (4)

CV
%
1.6
1.6
1.6
1.4

* repeatability = within-run precision
** intermediate precision = total precision / between run precision / between day precision

Method comparison
Cholesterol values for human serum and plasma samples obtained on a Roche/Hitachi cobas c 501 analyzer
(y) were compared with those determined using the same reagent on a Roche/Hitachi 917 analyzer (x).
Sample size (n) = 266
Passing/Bablok22
Linear regression
y = 1.002x + 0.045 mmol/L
y = 1.012x - 0.015 mmol/L
r = 0.997
τ = 0.953
The sample concentrations were between 1.53 and 18.5 mmol/L (59.1 and 715 mg/dL).

References
1. Greiling H, Gressner AM, eds. Lehrbuch der Klinischen Chemie und Pathobiochemie, 3rd ed.
Stuttgart/New York: Schattauer, 1995.
2. Liebermann C. Ber Dtsch chem Ges 1885;18:1803.
3. Burchard H. Beiträge zur Kenntnis der Cholesterine. Dissertation, Rostock 1889.
4. Abell L et al. Standard Methods in Clinical Chemistry 1958;26:2.
5. Allain CC et al. Clin Chem 1974;20:470.
6. Roeschlau P et al. Z Klin Chem Klin Biochem 1974;12:226.
7. Trinder P. Ann Clin Biochem 1969;6:24.
8. Siedel J, Hägele EO, Ziegenhorn J et al. Clin Chem 1983;29:1075.
9. Wiebe DA, Bernert JT. Clin Chem 1984;30:352.
10. Cohn JS, McNamara JR, Schaefer EJ. Lipoprotein Cholesterol Concentrations in the Plasma of Human
Subjects as Measured in the Fed and Fasted States. Clin Chem 1988;34:2456-2459.
11. Pisani T, Gebski CP, Leary ET et al. Accurate Direct Determination of Low-density Lipoprotein
Cholesterol Using an Immunoseparation Reagent and Enzymatic Cholesterol Assay. Arch Pathol Lab
Med 1995;119:1127.
12. Recommendations for Improving Cholesterol Measurement: A Report from the Laboratory
Standardization Panel of the National Cholesterol Education Program. NIH Publication No. 90-2964,
February 1990.
7 of 9 pages

Laboratory Name
Test Name: Cholesterol Gen.2
13. Nader R, Dufour DR, Cooper GR. Preanalytical Variation in Lipid, Lipoprotein, and Apolipoprotein
Testing. In: Rifai N, Warnick GR, and Dominiczak MH, editors. Handbook of Lipoprotein Testing. 2nd
ed. Washington: AACC press; p.176.
14. Tietz NW, ed. Clinical Guide to Laboratory Tests, 3rd ed. Philadelphia, PA: WB Saunders Company,
1995:130-131.
15. Use of Anticoagulants in Diagnostic Laboratory Investigations. WHO publication WHO/DIL/LAB/99.1
Rev. 2. Jan. 2002.
16. Siekmann L, Hüskes KP, Breuer H. 1976: Determination of cholesterol in serum using mass
fragmentography - a reference method in clinical chemistry. Z Anal Chem 279, 145-146.
17. Glick MR, Ryder KW, Jackson SA. Graphical Comparisons of Interferences in Clinical Chemistry
Instrumentation. Clin Chem 1986;32:470-475.
18. Breuer J. Report on the Symposium “Drug effects in Clinical Chemistry Methods”. Eur J Clin Chem
Clin Biochem 1996;34:385-386.
19. Sonntag O, Scholer A. Drug interference in clinical chemistry: recommendation of drugs and their
concentrations to be used in drug interference studies. Ann Clin Biochem 2001;38:376-385.
20. Study Group, European Atherosclerosis Society. Strategies for the prevention of coronary heart disease:
A policy statement of the European Atherosclerosis Society. European Heart Journal 1987;8:77.
21. Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection,
Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). NIH
Publication No 01-3670; May 2001.
22. Passing H, Bablok W et al. A General Regression Procedure for Method Transformation.
J Clin Chem Clin Biochem 1988;26:783-790.

Alternative method
_______________________________________________________________________________________
_______________________________________________________________________________________

FOR US CUSTOMERS ONLY: LIMITED WARRANTY
Roche Diagnostics warrants that this product will meet the specifications stated in the labeling when used in
accordance with such labeling and will be free from defects in material and workmanship until the expiration
date printed on the label. THIS LIMITED WARRANTY IS IN LIEU OF ANY OTHER WARRANTY,
EXPRESS OR IMPLIED, INCLUDING ANY IMPLIED WARRANTY OF MERCHANTABILITY OR
FITNESS FOR PARTICULAR PURPOSE. IN NO EVENT SHALL ROCHE DIAGNOSTICS BE LIABLE
FOR INCIDENTAL, INDIRECT, SPECIAL OR CONSEQUENTIAL DAMAGES.
COBAS, COBAS C, PRECINORM and PRECIPATH are trademarks of Roche. Other brand or product
names are trademarks of their respective holders.
© 2010, Roche Diagnostics
Version 7
2010-07

8 of 9 pages

Laboratory Name
Test Name: Cholesterol Gen.2

Reagent manufacturer
Roche Diagnostics GmbH, Sandhofer Strasse 116, D-68305 Mannheim
www.roche.com
Distribution in USA by:
Roche Diagnostics, Indianapolis, IN
US Customer Technical Support: 1-800-428-2336

Source document
Reagent Name: CHOL2
Package Insert Version: 2010-03, V7 English

9 of 9 pages

Application Sheet
Laboratory Name
Test Name: Creatinine Jaffé Gen.2

Order information
Creatinine Jaffé Gen.2
700 tests
Calibrator f.a.s. (12 x 3 mL)
Calibrator f.a.s. (12 x 3 mL,
for USA)
Precinorm U plus (10 x 3 mL)
Precinorm U plus (10 x 3 mL,
for USA)
Precipath U plus (10 x 3 mL)
Precipath U plus (10 x 3 mL,
for USA)
Precinorm U (20 x 5 mL)
Precipath U (20 x 5 mL)
Precinorm PUC (4 x 3 mL)
Precipath PUC (4 x 3 mL)
PreciControl ClinChem Multi 1
(20 x 5 mL)
PreciControl ClinChem Multi 1
(4 x 5 mL, for USA)
PreciControl ClinChem Multi 2
(20 x 5 mL)
PreciControl ClinChem Multi 2
(4 x 5 mL, for USA)
Diluent NaCl 9 % (50 mL)

y Indicates cobas c systems on which reagents can be used
Roche/Hitachi
cobas c systems
cobas c
cobas c
311
501/502
y
y
Cat. No. 04810716 190
System-ID 07 6928 2
Cat. No. 10759350 190
Code 401
Cat. No. 10759350 360
Code 401
Cat. No. 12149435 122
Cat. No. 12149435 160

Code 300
Code 300

Cat. No. 12149443 122
Cat. No. 12149443 160

Code 301
Code 301

Cat. No. 10171743
Cat. No. 10171778
Cat. No. 03121313
Cat. No. 03121291
Cat. No. 05117003

122
122
122
122
190

Code 300
Code 301
Code 240
Code 241
Code 391

Cat. No. 05947626 160

Code 391

Cat. No. 05117216 190

Code 392

Cat. No. 05947774 160

Code 392

Cat. No. 04489357 190

System-ID 07 6869 3

Effective date
Effective date for this procedure: ____________________________

Author
Source documentation compiled by Roche Diagnostics
Revised by: ___________________________________________

1 of 14 pages

Laboratory Name
Test Name: Creatinine Jaffé Gen.2

System information
For cobas c 311/501 analyzers:
CREJ2: ACN 690 (Rate blanked, compensated, serum and plasma)
CRJ2U: ACN 691 (Rate blanked, urine)
SCRE2: ACN 773 (STAT, compensated, serum and plasma, reaction time: 4)
SCR2U: ACN 774 (STAT, urine, reaction time: 4)
For cobas c 502 analyzer:
CREJ2: ACN 8690 (Rate blanked, compensated, serum and plasma)
CRJ2U: ACN 8691 (Rate blanked, urine)
SCRE2: ACN 8773 (STAT, compensated, serum and plasma, reaction time: 4)
SCR2U: ACN 8774 (STAT, urine, reaction time: 4)

Intended use
In vitro test for the quantitative determination of creatinine in human serum, plasma and urine on
Roche/Hitachi cobas c systems.

Summary1,2,3,4,5
Chronic kidney disease is a worldwide problem that carries a substantial risk for cardiovascular morbidity
and death. Current guidelines define chronic kidney disease as kidney damage or glomerular filtration rate
(GFR) less than 60 mL/min per 1.73 m2 for three months or more, regardless of cause.
The assay of creatinine in serum or plasma is the most commonly used test to assess renal function.
Creatinine is a break-down product of creatine phosphate in muscle, and is usually produced at a fairly
constant rate by the body (depending on muscle mass). It is freely filtered by the glomeruli and, under
normal conditions, is not re-absorbed by the tubules to any appreciable extent. A small but significant
amount is also actively secreted.
Since a rise in blood creatinine is observed only with marked damage of the nephrons, it is not suited to
detect early stage kidney disease. A considerably more sensitive test and better estimation of glomerular
filtration rate (GFR) is given by the creatinine clearance test based on creatinine’s concentration in urine and
serum or plasma, and urine flow rate. For this test a precisely timed urine collection (usually 24 hours) and a
blood sample are needed. However, since this test is prone to error due to the inconvenient collection of
timed urine, mathematical attempts to estimate GFR based only on the creatinine concentration in serum or
plasma have been made. Among the various approaches suggested, two have found wide recognition: that of
Cockroft and Gault and that based on the results of the MDRD trial. While the first equation was derived
from data obtained with the conventional Jaffé method, a newer version of the second is usable for IDMStraceable creatinine methods. Both are applicable for adults. In children, the Bedside Schwartz formula
should be used.6,7,8,9

2 of 14 pages

Laboratory Name
Test Name: Creatinine Jaffé Gen.2
In addition to the diagnosis and treatment of renal disease, the monitoring of renal dialysis, creatinine
measurements are used for the calculation of the fractional excretion of other urine analytes (e. g., albumin,
α-amylase). Numerous methods were described for determining creatinine. Automated assays established in
the routine laboratory include the Jaffé alkaline picrate method in various modifications, as well as
enzymatic tests.

Test principle10,11,12
This kinetic colorimetric assay is based on the Jaffé method. In alkaline solution, creatinine forms a yelloworange complex with picrate. The rate of dye formation is proportional to the creatinine concentration in the
specimen. The assay uses “rate-blanking” to minimize interference by bilirubin. To correct for non-specific
reaction caused by serum/plasma pseudo-creatinine chromogens, including proteins and ketones, the results
for serum or plasma are corrected by -26 µmol/L (-0.3 mg/dL).
Alkaline pH

Creatinine + picric acid

yellow-orange complex

Reagents - working solutions
R1
R2/R3

Potassium hydroxide: 900 mmol/L; phosphate: 135 mmol/L; pH ≥ 13.5; preservative;
stabilizer
Picric acid: 38 mmol/L; pH 6.5; non reactive buffer

Precautions and warnings
For in vitro diagnostic use.
Exercise the normal precautions required for handling all laboratory reagents.
Safety data sheet available for professional user on request.
Disposal of all waste material should be in accordance with local guidelines.
This kit contains components classified as follows according to the European directive 1999/45/EC.
C – Corrosive. R1 contains potassium hydroxide.
R 1: Explosive when dry. R 4: Forms very sensitive, explosive metallic compounds. R 34: Causes burns.
S 24-25: Avoid contact with skin and eyes. S 26: In case of contact with eyes, rinse immediately with plenty
of water and seek medical advice. S 35: This material and its container must be disposed of in a safe way.
S 36/37/39: Wear suitable protective clothing, gloves and eye/face protection.
S 45: In case of accident or if you feel unwell, seek medical advice immediately (show the label where
possible).
Contact phone: all countries: +49-621-7590, USA: +1-800-428-2336

Reagent handling
Ready for use.

3 of 14 pages

Laboratory Name
Test Name: Creatinine Jaffé Gen.2

Storage and stability
CREJ2
Shelf life at 15-25 °C:
On-board in use and refrigerated on the analyzer:

See expiration date on cobas c pack label.
8 weeks

Diluent NaCl 9 %
Shelf life at 2-8 °C:
On-board in use and refrigerated on the analyzer:

See expiration date on cobas c pack label.
12 weeks

Specimen collection and preparation13
For specimen collection and preparation, only use suitable tubes or collection containers.
Only the specimens listed below were tested and found acceptable.
Serum.
Plasma: Li-heparin and K2-EDTA plasma.
The sample types listed were tested with a selection of sample collection tubes that were commercially
available at the time of testing, i.e. not all available tubes of all manufacturers were tested. Sample collection
systems from various manufacturers may contain differing materials which could affect the test results in
some cases. When processing samples in primary tubes (sample collection systems), follow the instructions
of the tube manufacturer.
Urine.
Collect urine without using additives. If urine must be collected with a preservative for other analytes, only
hydrochloric acid (14 to 47 mmol/L urine, e.g. 5 mL 10 % HCl or 5 mL 30 % HCl per liter urine) or boric
acid (81 mmol/L, e.g. 5 g per liter urine) may be used.
Stability in serum/plasma:14
7 days at 15-25 °C
7 days at 2-8 °C
3 months at (-15)-(-25) °C
Stability in urine (without preservative):14

2 days at 15-25 °C
6 days at 2-8 °C
6 months at (-15)-(-25) °C

Stability in urine (with preservative):15

3 days at 15-25 °C
8 days at 2-8 °C
3 weeks at (-15)-(-25) °C

Centrifuge samples containing precipitates before performing the assay.

Materials provided
See “Reagents - working solutions” section for reagents.

4 of 14 pages

Laboratory Name
Test Name: Creatinine Jaffé Gen.2

Assay
For optimum performance of the assay, follow the directions given in this document for the analyzer
concerned. Refer to the appropriate operator's manual for analyzer-specific assay instructions.
The performance of applications not validated by Roche is not warranted and must be defined by the user.

Application for serum and plasma
cobas c 311 test definition
Assay type
Reaction time / Assay points
Wavelength (sub/main)
Reaction direction
Units
Reagent pipetting
R1
R3

Rate A
10 / 27-37 - 15-23
(STAT 4 / 12-19)
570/505 nm
Increase
µmol/L (mg/dL, mmol/L)
Diluent (H2O)
13 µL
77 µL
17 µL
30 µL

Sample volumes

Sample

Normal
Decreased
Increased

10 µL
10 µL
10 µL

Sample
–
20 µL
–

Sample dilution
Diluent (NaCl)
–
80 µL
–

Enter the correction value for the non-specific protein reaction as the instrument factor y = ax + b for mg/dL
or for µmol/L, where a = 1.0 and b = -0.3 (mg/dL) or a = 1.0 and b = -26 (µmol/L).
cobas c 501/502 test definition
Assay type
Rate A
Reaction time / Assay points
10 / 42-52 - 24-34
(STAT 4 / 17-27)
Wavelength (sub/main)
570/505 nm
Reaction direction
Increase
Units
µmol/L (mg/dL, mmol/L)
Reagent pipetting
Diluent (H2O)
R1
13 µL
77 µL
R3
17 µL
30 µL
5 of 14 pages

Laboratory Name
Test Name: Creatinine Jaffé Gen.2

Sample volumes

Sample

Normal
Decreased
Increased

10 µL
10 µL
10 µL

Sample
–
20 µL
–

Sample dilution
Diluent (NaCl)
–
80 µL
–

Enter the correction value for the non-specific protein reaction as the instrument factor y = ax + b for mg/dL
or for µmol/L, where a = 1.0 and b = -0.3 (mg/dL) or a = 1.0 and b = -26 (µmol/L).

Application for urine
cobas c 311 test definition
Assay type
Reaction time / Assay points
Wavelength (sub/main)
Reaction direction
Units
Reagent pipetting
R1
R3

Rate A
10 / 27-37 - 15-23
(STAT 4 / 12-19)
570/505 nm
Increase
µmol/L (mg/dL, mmol/L)
Diluent (H2O)
13 µL
77 µL
17 µL
30 µL

Sample volumes

Sample

Normal
Decreased
Increased

10 µL
10 µL
10 µL

cobas c 501/502 test definition
Assay type
Reaction time / Assay points

Sample
6 µL
2 µL
10 µL

Wavelength (sub/main)
Reaction direction
Units
Reagent pipetting
R1
R3

Rate A
10 / 42-52 - 24-34
(STAT 4 / 17-27)
570/505 nm
Increase
µmol/L (mg/dL, mmol/L)
Diluent (H2O)
13 µL
77 µL
17 µL
30 µL

Sample volumes

Sample

Normal
Decreased
Increased

10 µL
10 µL
10 µL

Sample
6 µL
2 µL
10 µL
6 of 14 pages

Sample dilution
Diluent (NaCl)
144 µL
180 µL
115 µL

Laboratory Name
Test Name: Creatinine Jaffé Gen.2

Calibration
Calibrators
Calibration mode
Calibration frequency

S1: H2O
S2: C.f.a.s.
Linear
2-point calibration
• after reagent lot change
• as required following quality control procedures

Traceability: This method has been standardized against ID/MS.
For the USA, this method has been standardized against a primary reference material (SRM 914 and
SRM 967 (ID/MS)).

Quality control
For quality control, use control materials as listed in the “Order information” section.
Other suitable control material can be used in addition.
Serum/plasma
For quality control use undiluted serum control material as listed above. Other suitable control material can
be used in addition.
Urine
For quality control use Precinorm PUC and Precipath PUC as listed above. Other suitable control material
can be used in addition.
The control intervals and limits should be adapted to each laboratory’s individual requirements. Values
obtained should fall within the defined limits. Each laboratory should establish corrective measures to be
taken if values fall outside the limits.
Follow the applicable government regulations and local guidelines for quality control.
If controls do not recover within the specified limits, take the following corrective action:
_______________________________________________________________________________________
_______________________________________________________________________________________

Calculation
Roche/Hitachi cobas c systems automatically calculate the analyte concentration of each sample.
Conversion factors:
µmol/L x 0.0113 = mg/dL
µmol/L x 0.001 = mmol/L

Limitations – interference
Criterion: Recovery within ± 10 % of initial value at a creatinine concentration of 80 µmol/L (0.90 mg/dL) in
serum/plasma and 2500 µmol/L (28.3 mg/dL) in urine.
Serum/plasma
Icterus (CREJ2):16 No significant interference up to an I index of 5 for conjugated bilirubin and 10 for
unconjugated bilirubin (approximate conjugated bilirubin concentration: 86 µmol/L (5 mg/dL) and
approximate unconjugated bilirubin concentration: 171 µmol/L (10 mg/dL)).
Icterus (SCRE2):16 No significant interference up to an I index of 2 for conjugated bilirubin and 3 for
unconjugated bilirubin (approximate conjugated bilirubin concentration: 34 µmol/L (2 mg/dL) and
approximate unconjugated bilirubin concentration: 51 µmol/L (3 mg/dL)).
7 of 14 pages

Laboratory Name
Test Name: Creatinine Jaffé Gen.2
Hemolysis:16 No significant interference up to an H index of 1000 (approximate hemoglobin concentration:
621 µmol/L (1000 mg/dL)).
Lipemia (Intralipid):16 No significant interference up to an L index of 800. There is poor correlation between
the L index (corresponds to turbidity) and triglycerides concentration.
Drugs: No interference was found at therapeutic levels using common drug panels.17,18
Exception: Cefoxitin causes artificially high creatinine results.
Exception: Cyanokit (Hydroxocobalamin) may cause interference with results.
Values < 15 µmol/L (< 0.17 mg/dL) or negative results are reported in rare cases in children < 3 years and in
elderly patients. In such cases use the Creatinine plus test to assay the sample.
Do not use Creatinine Jaffé for the testing of creatinine in hemolyzed samples from neonates, infants or
adults with HbF levels ≥ 60 mg/dL for CREJ2 applications (≥ 30 mg/dL for SCRE2 applications).19 In such
cases, use the Creatinine plus test (≤ 600 mg/dL HbF) to assay the sample.
Estimation of the Glomerular Filtration Rate (GFR) on the basis of the Schwartz Formula can lead to an
overestimation.20
In very rare cases, gammopathy, in particular type IgM (Waldenström's macroglobulinemia), may cause
unreliable results.
The presence of ketone bodies can cause artificially high results in serum and plasma.
Urine
Icterus: No significant interference up to a conjugated bilirubin concentration of 855 µmol/L (50 mg/dL).
Hemolysis: No significant interference up to a hemoglobin concentration of 621 µmol/L (1000 mg/dL).
Glucose < 120 mmol/L (< 2162 mg/dL) and urobilinogen < 676 µmol/L (< 40 mg/dL) do not interfere.
Drugs: No interference was found at therapeutic levels using common drug panels.18
Exception: Cyanokit (Hydroxocobalamin) may cause interference with results.
High homogentisic acid concentrations in urine samples lead to false results.
The presence of ketone bodies can cause artificially high results in urine.
For diagnostic purposes, the results should always be assessed in conjunction with the patient's medical
history, clinical examination and other findings.
ACTION REQUIRED
Special Wash Programming: The use of special wash steps is mandatory when certain test combinations are
run together on Roche/Hitachi cobas c systems. The latest version of the Carry over evasion list can be
found with the NaOHD/SMS/Multiclean/SCCS or the NaOHD/SMS/SmpCln1 + 2/SCCS Method Sheets.
For further instructions refer to the operator manual.
cobas c 502 analyzer: All special wash programming necessary for avoiding carry over is available via the
cobas link, manual input is not required.
Where required, special wash/carry over evasion programming must be implemented prior to
reporting results with this test.

Limits and ranges
Measuring range
Serum/plasma
15-2200 µmol/L (0.17-24.9 mg/dL)
The technical limit in the instrument setting is defined as 0.47-25.2 mg/dL due to the compensation factor of
0.3.
Determine samples having higher concentrations via the rerun function. Dilution of samples via the rerun
function is a 1:5 dilution. Results from samples diluted by the rerun function are automatically multiplied by
a factor of 5.

8 of 14 pages

Laboratory Name
Test Name: Creatinine Jaffé Gen.2
Urine
375-55000 µmol/L (4.2-622 mg/dL)
Determine samples having higher concentrations via the rerun function. Dilution of samples via the rerun
function is a 1:3.6 dilution. Results from samples diluted by the rerun function are automatically multiplied
by a factor of 3.6.
Lower limits of measurement
Lower detection limit of the test
Serum/plasma
15 µmol/L (0.17 mg/dL)
The lower detection limit represents the lowest measurable analyte level that can be distinguished from zero.
It is calculated as the value lying three standard deviations above that of the lowest standard
(standard 1 + 3 SD, repeatability, n = 21).
Urine
375 µmol/L (4.2 mg/dL)
The lower detection limit represents the lowest measurable analyte level that can be distinguished from zero.
It is calculated as the value lying three standard deviations above that of the lowest standard
(standard 1 + 3 SD, repeatability, n = 21).

Expected values
Serum/plasma
Adults21
Females
Males
Children22
Neonates (premature)
Neonates (full term)
2-12 m
1- < 3 y
3- < 5 y
5- < 7 y
7- < 9 y
9- < 11 y
11- < 13 y
13- < 15 y
Urine
1st morning urine21
Females
Males
24-hour urine23
Females
Males
Creatinine clearance23,24

44-80 µmol/L
62-106 µmol/L

(0.50-0.90 mg/dL)
(0.70-1.20 mg/dL)

25-91 µmol/L
21-75 µmol/L
15-37 µmol/L
21-36 µmol/L
27-42 µmol/L
28-52 µmol/L
35-53 µmol/L
34-65 µmol/L
46-70 µmol/L
50-77 µmol/L

(0.29-1.04 mg/dL)
(0.24-0.85 mg/dL)
(0.17-0.42 mg/dL)
(0.24-0.41 mg/dL)
(0.31-0.47 mg/dL)
(0.32-0.59 mg/dL)
(0.40-0.60 mg/dL)
(0.39-0.73 mg/dL)
(0.53-0.79 mg/dL)
(0.57-0.87 mg/dL)

2470-19200 µmol/L
3450-22900 µmol/L

(28-217 mg/dL)
(39-259 mg/dL)

7000-14000 µmol/24 h
9000-21000 µmol/24 h

(740-1570 mg/24 h)
(1040-2350 mg/24 h)

71-151 mL/min
9 of 14 pages

Laboratory Name
Test Name: Creatinine Jaffé Gen.2
Refer to reference 25 for a prospective study on creatinine clearance in children.25
Roche has not evaluated reference ranges in a pediatric population.
Each laboratory should investigate the transferability of the expected values to its own patient population and
if necessary determine its own reference ranges.

Specific performance data
Representative performance data on the analyzers are given below. Results obtained in individual
laboratories may differ.

Precision
Precision was determined using human samples and controls in an internal protocol. Serum/plasma:
repeatability* (n = 21), intermediate precision** (3 aliquots per run, 1 run per day, 21 days); Urine:
repeatability* (n = 21), intermediate precision** (3 aliquots per run, 1 run per day, 10 days). The following
results were obtained:
Serum/plasma (CREJ2)
Repeatability*
Mean
SD
CV
µmol/L (mg/dL)
µmol/L (mg/dL)
%
Precinorm U
105 (1.19)
2 (0.03)
2.1
Precipath U
360 (4.07)
4 (0.05)
1.1
Human serum 1
206 (2.33)
3 (0.03)
1.2
Human serum 2
422 (4.77)
5 (0.06)
1.3
Intermediate precision**
Precinorm U
Precipath U
Human serum 3
Human serum 4
Urine (CRJ2U)
Repeatability*
Control Level 1
Control Level 2
Human urine 1
Human urine 2
Intermediate precision**
Control Level 1
Control Level 2
Human urine 3
Human urine 4

Mean
µmol/L (mg/dL)
101 (1.14)
351 (3.97)
201 (2.27)
411 (4.64)

SD
µmol/L (mg/dL)
4 (0.05)
8 (0.09)
5 (0.06)
9 (0.10)

CV
%
3.5
2.2
2.5
2.2

Mean
µmol/L (mg/dL)
8083 (91.3)
15618 (177)
19318 (218)
7958 (89.9)

SD
µmol/L (mg/dL)
115 (1.3)
213 (2)
234 (3)
130 (1.5)

CV
%
1.4
1.4
1.2
1.6

Mean
µmol/L (mg/dL)
8130 (91.9)
15533 (176)
19353 (219)
7932 (89.6)

SD
µmol/L (mg/dL)
164 (1.9)
251 (3)
385 (4)
166 (1.9)

CV
%
2.0
1.6
2.0
2.1

10 of 14 pages

Laboratory Name
Test Name: Creatinine Jaffé Gen.2
Serum/plasma (SCRE2)
Repeatability*
Precinorm U
Precipath U
Human serum 1
Human serum 2
Intermediate precision**
Precinorm U
Precipath U
Human serum 3
Human serum 4
Urine (SCR2U)
Repeatability*
Control Level 1
Control Level 2
Human urine 1
Human urine 2
Intermediate precision**
Control Level 1
Control Level 2
Human urine 3
Human urine 4

Mean
µmol/L (mg/dL)
106 (1.20)
346 (3.91)
543 (6.14)
69 (0.78)

SD
µmol/L (mg/dL)
2 (0.02)
5 (0.06)
6 (0.07)
2 (0.02)

CV
%
2.2
1.5
1.1
3.1

Mean
µmol/L (mg/dL)
100 (1.13)
334 (3.77)
522 (5.90)
64 (0.72)

SD
µmol/L (mg/dL)
4 (0.05)
10 (0.11)
12 (0.14)
3 (0.03)

CV
%
4.0
3.0
2.4
5.0

Mean
µmol/L (mg/dL)
6287 (71.0)
15252 (172)
24174 (273)
2146 (24.2)

SD
µmol/L (mg/dL)
82 (0.9)
182 (2)
212 (2)
48 (0.5)

CV
%
1.2
1.2
0.9
2.2

Mean
µmol/L (mg/dL)
6943 (78.5)
15394 (174)
24230 (274)
2184 (24.7)

SD
µmol/L (mg/dL)
114 (1.3)
229 (3)
354 (4)
54 (0.6)

CV
%
1.6
1.5
1.5
2.5

* repeatability = within-run precision
** intermediate precision = total precision / between run precision / between day precision

Method comparison
Creatinine values for human serum, plasma and urine samples obtained on a Roche/Hitachi cobas c 501
analyzer (y) were compared with those determined on Roche/Hitachi 917/MODULAR P analyzers (x), using
the corresponding Roche/Hitachi reagent.
Serum/plasma (CREJ2)
Sample size (n) = 273
Passing/Bablok26
Linear regression
y = 1.000x - 0.653 µmol/L
y = 1.002x - 0.978 µmol/L
r = 0.999
τ = 0.973
The sample concentrations were between 38 and 2178 µmol/L (0.429 and 24.6 mg/dL).

11 of 14 pages

Laboratory Name
Test Name: Creatinine Jaffé Gen.2
Urine (CRJ2U)
Sample size (n) = 223
Passing/Bablok26
Linear regression
y = 0.999x + 20.7 µmol/L
y = 0.999x + 41.5 µmol/L
r = 0.999
τ = 0.969
The sample concentrations were between 934 and 50228 µmol/L (10.6 and 568 mg/dL).
Serum/plasma (SCRE2)
Sample size (n) = 224
Passing/Bablok26
Linear regression
y = 1.000x - 14.4 µmol/L
y = 0.996x - 12.2 µmol/L
r = 0.999
τ = 0.964
The sample concentrations were between 66 and 1775 µmol/L (0.746 and 20.1 mg/dL).
Urine (SCR2U)
Sample size (n) = 223
Passing/Bablok26
Linear regression
y = 0.999x + 67.8 µmol/L
y = 0.998x + 113 µmol/L
r = 0.999
τ = 0.973
The sample concentrations were between 931 and 48729 µmol/L (10.5 and 551 mg/dL).

References
1. Thomas C, Thomas L. Labordiagnostik von Erkrankungen der Nieren und ableitenden Harnwege. In:
Thomas L, ed. Labor und Diagnose. 6th ed. Frankfurt/Main: TH-Books 2005;520-85.
2. Lamb E, Newman DJ, Price CP. Kidney function tests. In: Burtis CA, Ashwood ER, Bruns DE. Tietz
textbook of clinical chemistry and molecular diagnostics. 4th ed. St. Louis, MO: Elsevier Saunders
2006;797-835.
3. http://www.kidney.org/
4. http://www.nkdep.nih.gov/
5. Lamb EJ, Tomson CRV, Roderick PJ. Estimating kidney function in adults using formulae. Ann Clin
Biochem 2005;42:321-45.
6. Miller WG. Editorial on Estimating glomerular filtration rate. Clin Chem Lab Med 2009;47(9):1017–
1019.
7. Schwartz GJ, Muñoz A, Schneider MF et al. New Equations to Estimate GFR in Children with CKD. J
Am Soc Nephrol 2009;20:629–637.
8. Schwartz GJ, Work DF. Measurement and Estimation of GFR in Children and Adolescents. Clin J Am
Soc Nephrol 2009;4:1832–1843.
9. Staples A, LeBlond R, Watkins S et al. Validation of the revised Schwartz estimating equation in a
predominantly non-CKD population. Pediatr Nephrol 2010 Jul 22;25:2321–2326.
10. Jaffé M. Über den Niederschlag, welchen Pikrinsäure in normalem Harn erzeugt und über eine neue
Reaktion des Kreatinins. Z Physiol Chem 1886;10:391-400.
11. Fabiny DL, Ertinghausen G. Automated reaction-rate method for determination of serum creatinine with
the CentrifiChem. Clin Chem 1971;17:696-700.
12. Bartels H, Böhmer M. Micro-determination of creatinine. Clin Chim Acta 1971;32:81-85.
13. Guder WG, Narayanan S, Wisser H et al. List of Analytes; Pre-analytical Variables. Brochure in:
Samples: From the Patient to the Laboratory. Darmstadt: GIT Verlag 1996.
14. Guder WG, da Fonseca-Wollheim F, Ehret W et al. Die Qualität diagnostischer Proben, 6. Aufl.
Heidelberg: BD Diagnostics, 2009.
15. Data on file at Roche Diagnostics.
12 of 14 pages

Laboratory Name
Test Name: Creatinine Jaffé Gen.2
16. Glick MR, Ryder KW, Jackson SA. Graphical Comparisons of Interferences in Clinical Chemistry
Instrumentation. Clin Chem 1986;32:470-475.
17. Breuer J. Report on the Symposium “Drug effects in Clinical Chemistry Methods”. Eur J Clin Chem
Clin Biochem 1996;34:385-386.
18. Sonntag O, Scholer A. Drug interference in clinical chemistry: recommendation of drugs and their
concentrations to be used in drug interference studies. Ann Clin Biochem 2001: 38:376–385.
19. Mazzachi BC, Phillips JW, Peake MJ. Is the Jaffé creatinine assay suitable for neonates? Clin Biochem
Revs 1998;19:82.
20. Filler G, Priem F, Lepage N et al. β-Trace Protein, Cystatin C, β2-Microglobulin, and Creatinine
Compared for Detecting Impaired Glomerular Filtration Rates in Children. Clin Chem 2002;48:729-736.
21. Mazzachi BC, Peake MJ, Ehrhardt V. Reference Range and Method Comparison Studies for Enzymatic
and Jaffé Creatinine Assays in Plasma and Serum and Early Morning Urine. Clin Lab 2000;46:53-55.
22. Schlebusch H, Liappis N, Kalina E et al. High sensitive CRP and creatinine: reference intervals from
infancy to childhood. J Lab Med 2002;26:341-346.
23. Junge W, Wilke B, Halabi A et al. Determination of reference intervals for serum creatinine, creatinine
excretion and creatinine clearance with an enzymatic and a modified Jaffé method. Clin Chim Acta
2004;344:137-148.
24. Zawta B, Delanghe J, Taes Y et al. Arithmetic Compensation for Pseudo-Creatinine Interferences of the
Creatinine Jaffé Method and its Effect on Creatinine Clearance Results. Clin Chem Part 2, Suppl S June
2001;46(6):487.
25. Wuyts B, Bernard D, van den Noortgate N, van de Walle J et al. Reevaluation of Formulas for Predicting
Creatinine Clearance in Adults and Children Using Compensated Creatinine Methods. Clin Chem
2003;49:1011-1014.
26. Passing H, Bablok W, Bender R, et al. A General Regression Procedure for Method Transformation.
J Clin Chem Clin Biochem 1988;26:783-790.

Alternative method
_______________________________________________________________________________________
_______________________________________________________________________________________

13 of 14 pages

Laboratory Name
Test Name: Creatinine Jaffé Gen.2

Source document
Reagent Name: CREJ2
Package Insert Version: 2011-07, V13 English

14 of 14 pages

Application Sheet
Laboratory Name
Test Name: Triglycerides
y Indicates cobas c systems on which reagents can be used
Order information

Triglycerides
250 tests
Calibrator f.a.s. (12 x 3 mL)
Calibrator f.a.s. (12 x 3 mL,
for USA)
Precinorm U plus (10 x 3 mL)
Precinorm U plus (10 x 3 mL,
for USA)
Precipath U plus (10 x 3 mL)
Precipath U plus (10 x 3 mL,
for USA)
Precinorm U (20 x 5 mL)
Precipath U (20 x 5 mL)
Precinorm L (4 x 3 mL)
Precipath L (4 x 3 mL)
Diluent NaCl 9 % (50 mL)

Cat. No. 20767107 322
Cat. No. 10759350 190
Cat. No. 10759350 360

System-ID 07 6710 7
Code 401
Code 401

Cat. No. 12149435 122
Cat. No. 12149435 160

Code 300
Code 300

Cat. No. 12149443 122
Cat. No. 12149443 160

Code 301
Code 301

Cat. No. 10171743 122
Cat. No. 10171778 122
Cat. No. 10781827 122
Cat. No. 11285874 122
Cat. No. 04489357 190

Code 300
Code 301
Code 304
Code 305
System-ID 07 6869 3

Effective date
Effective date for this procedure: ____________________________

Author
Source documentation compiled by Roche Diagnostics
Revised by: ___________________________________________

System information
For cobas c 311/501 analyzers:
TRIGL: ACN 781
For cobas c 502 analyzer:
TRIGL: ACN 8781

1 of 8 pages

Roche/Hitachi
cobas c systems
cobas c
cobas c
311
501/502
y
y

Laboratory Name
Test Name: Triglycerides

Intended use
In vitro test for the quantitative determination of triglycerides in human serum and plasma on Roche/Hitachi
cobas c systems.

Summary1,2,3,4,5,6
Triglycerides are esters of the trihydric alcohol glycerol with 3 long-chain fatty acids. They are partly
synthesized in the liver and partly ingested in food.
The determination of triglycerides is utilized in the diagnosis and treatment of patients having diabetes
mellitus, nephrosis, liver obstruction, lipid metabolism disorders and numerous other endocrine diseases.
The enzymatic triglycerides assay as described by Eggstein and Kreutz still required saponification with
potassium hydroxide. Numerous attempts were subsequently made to replace alkaline saponification by
enzymatic hydrolysis with lipase. Bucolo and David tested a lipase/protease mixture; Wahlefeld used an
esterase from the liver in combination with a particularly effective lipase from Rhizopus arrhizus for
hydrolysis.
This method is based on the work by Wahlefeld using a lipoprotein lipase from microorganisms for the rapid
and complete hydrolysis of triglycerides to glycerol followed by oxidation to dihydroxyacetone phosphate
and hydrogen peroxide. The hydrogen peroxide produced then reacts with 4-aminophenazone and 4chlorophenol under the catalytic action of peroxidase to form a red dyestuff (Trinder endpoint reaction). The
color intensity of the red dyestuff formed is directly proportional to the triglyceride concentration and can be
measured photometrically.

Test principle6
Enzymatic colorimetric test.
LPL

triglycerides + 3 H2O

glycerol + 3 RCOOH
GK

glycerol + ATP

glycerol-3-phosphate + ADP
2+
Mg

GPO

glycerol-3-phosphate + O2

dihydroxyacetone phosphate + H2O2
peroxidase

H2O2 + 4-aminophenazone + 4-chlorophenol

4-(p-benzoquinone-monoimino)-phenazone
+ 2 H2O + HCl

Reagents - working solutions
R1 PIPES buffer: 50 mmol/L, pH 6.8; Mg2+: 40 mmol/L; sodium cholate: 0.20 mmol/L; ATP:
≥ 1.4 mmol/L; 4-aminophenazone: ≥ 0.13 mmol/L; 4-chlorophenol: 4.7 mmol/L; lipoprotein lipase
(Pseudomonas spec.): ≥ 83 µkat/L; glycerokinase (Bacillus stearothermophilus): ≥ 3 µkat/L; glycerol
phosphate oxidase (E. coli): ≥ 41 µkat/L; peroxidase (horseradish): ≥ 1.6 µkat/L; preservative

2 of 8 pages

Laboratory Name
Test Name: Triglycerides

Reagent handling
Ready for use.

Storage and stability
TRIGL
Shelf life at 2-8 °C:
On-board in use and refrigerated on the analyzer:

See expiration date on cobas c pack label.
8 weeks

Diluent NaCl 9 %
Shelf life at 2-8 °C:
On-board in use and refrigerated on the analyzer:

See expiration date on cobas c pack label.
12 weeks

Specimen collection and preparation
For specimen collection and preparation, only use suitable tubes or collection containers.
Only the specimens listed below were tested and found acceptable.
Serum.
Plasma: Li-heparin and K2-EDTA plasma
The sample types listed were tested with a selection of sample collection tubes that were commercially
available at the time of testing, i.e. not all available tubes of all manufacturers were tested. Sample collection
systems from various manufacturers may contain differing materials which could affect the test results in
some cases. When processing samples in primary tubes (sample collection systems), follow the instructions
of the tube manufacturer.
Centrifuge samples containing precipitates before performing the assay.
Stability:7
5-7 days at 2-8 °C
3 months at (-15)-(-25) °C
several years at (-60)-(-80) °C

Materials provided
See “Reagents - working solutions” section for reagents.

Laboratory Name
Test Name: Triglycerides

Application for serum and plasma
cobas c 311 test definition
Assay type
Reaction time / Assay points
Wavelength (sub/main)
Reaction direction
Units
Reagent pipetting
R1
Sample volumes

Diluent (H2O)
28 µL

Sample
–
15 µL
–

2 µL
4 µL
4 µL

cobas c 501/502 test definition
Assay type
Reaction time / Assay points
Wavelength (sub/main)
Reaction direction
Units
Reagent pipetting
R1

Normal
Decreased
Increased

120 µL
Sample

Normal
Decreased
Increased

Sample volumes

1 Point
10 / 57
700/505 nm
Increase
mmol/L (mg/dL, g/L)

Sample dilution
Diluent (NaCl)
–
135 µL
–

1 Point
10 / 70
700/505 nm
Increase
mmol/L (mg/dL, g/L)
120 µL

Diluent (H2O)
28 µL

Sample
Sample
–
15 µL
–

2 µL
4 µL
4 µL

Sample dilution
Diluent (NaCl)
–
135 µL
–

Calibration
Calibrators
Calibration mode
Calibration frequency

S1: H2O
S2: C.f.a.s.
Linear
2-point calibration
• after reagent lot change
• and as required following quality control procedures

Traceability: This method has been standardized against the ID/MS method.

4 of 8 pages

Laboratory Name
Test Name: Triglycerides

Quality control
For quality control, use control materials as listed in the “Order information” section.
Other suitable control material can be used in addition.
The control intervals and limits should be adapted to each laboratory’s individual requirements. Values
obtained should fall within the defined limits. Each laboratory should establish corrective measures to be
taken if values fall outside the limits.
Follow the applicable government regulations and local guidelines for quality control.
If controls do not recover within the specified limits, take the following corrective action:
_______________________________________________________________________________________
_______________________________________________________________________________________

Calculation
Roche/Hitachi cobas c systems automatically calculate the analyte concentration of each sample.
Conversion factors:

mmol/L x 88.5 = mg/dL
mg/dL x 0.0113 = mmol/L

Limitations - interference8
Criterion: Recovery within ± 10 % of initial values at triglyceride levels of 2.3 mmol/L (203 mg/dL).
Icterus: No significant interference up to an I index of 10 for conjugated and 35 for unconjugated bilirubin
(approximate conjugated bilirubin concentration: 171 µmol/L (10 mg/dL) and approximate unconjugated
bilirubin concentration: 599 µmol/L (35 mg/dL)).
Hemolysis: No significant interference up to an H index of 700 (approximate hemoglobin concentration:
434 µmol/L (700 mg/dL)).
Lipemia: The L index correlates with sample turbidity but not with triglycerides level. Extremely lipemic
samples (triglycerides greater than 3000 mg/dL) can produce normal results.9
Prozone Check: The flag > Kin is an indicator for extremely high triglyceride concentrations in the sample.
False normal results are due to oxygen depletion during assay reaction.
Endogenous unesterified glycerol in the sample will falsely elevate serum triglycerides.
Drugs: No interference was found at therapeutic concentrations using common drug panels.10,11
Exception: Ascorbic acid and calcium dobesilate cause artificially low triglyceride results. Intralipid is
directly measured as analyte in this assay and leads to high triglyceride results.
In very rare cases, gammopathy, in particular type IgM (Waldenström's macroglobulinemia), may cause
unreliable results.
For diagnostic purposes, the results should always be assessed in conjunction with the patient's medical
history, clinical examination and other findings.
ACTION REQUIRED
Special Wash Programming: The use of special wash steps is mandatory when certain test combinations are
run together on Roche/Hitachi cobas c systems. The latest version of the Carry over evasion list can be
found with the NaOHD/SMS/Multiclean/SCCS or the NaOHD/SMS/SmpCln1 + 2/SCCS Method Sheets.
For further instructions refer to the operator manual.
cobas c 502 analyzer: All special wash programming necessary for avoiding carry over is available via the
cobas link, manual input is not required.
Where required, special wash/carry over evasion programming must be implemented prior to
reporting results with this test.

5 of 8 pages

Laboratory Name
Test Name: Triglycerides

Limits and ranges
Measuring range
0.1-10.0 mmol/L (8.85-885 mg/dL)
Determine samples having higher concentrations via the rerun function. Recommended dilution of samples
via the rerun functions is a 1:5 dilution. Results from samples diluted by the rerun function are automatically
multiplied by a factor of 5.
Lower limits of measurement
Lower detection limit of the test
0.1 mmol/L (8.85 mg/dL)
The lower detection limit represents the lowest measurable analyte level that can be distinguished from zero.
It is calculated as the value lying three standard deviations above that of the lowest standard (standard
1 + 3 SD, repeatability, n = 21).

Expected values according to NCEP12
Normal range: < 2.26 mmol/L (< 200 mg/dL)
Clinical interpretation according to the recommendations of the European Atherosclerosis Society:13
Cholesterol
Triglycerides
Cholesterol
Cholesterol
Triglycerides

mmol/L
< 5.18
< 2.26

mg/dL
< 200
< 200

Lipid metabolism disorder

5.18-7.77

200-300

Yes
if HDL-cholesterol
< 0.9 mmol/L (< 35 mg/dL)

> 7.77
> 2.26

> 300
> 200

Yes

Note: If the free glycerol is to be taken into account, then 0.11 mmol/L (10 mg/dL) must be subtracted from
the triglycerides value obtained.7
Each laboratory should investigate the transferability of the expected values to its own patient population and
if necessary determine its own reference ranges.

Specific performance data
Representative performance data on the analyzers are given below. Results obtained in individual
laboratories may differ.

Precision
Precision was determined using human samples and controls in an internal protocol. Repeatability* (n = 21),
intermediate precision** (3 aliquots per run, 1 run per day, 21 days). The following results were obtained:
Repeatability*
Mean
SD
CV
mmol/L (mg/dL)
mmol/L (mg/dL)
%
Precinorm U
1.41 (125)
0.01 (1)
0.9
Precipath U
2.40 (212)
0.02 (2)
0.8
Human serum 1
1.67 (148)
0.02 (2)
1.1
Human serum 2
2.72 (241)
0.02 (2)
0.7

6 of 8 pages

Laboratory Name
Test Name: Triglycerides
Intermediate precision**
Precinorm U
Precipath U
Human serum 3
Human serum 4

Mean
mmol/L (mg/dL)
1.39 (123)
2.33 (206)
1.18 (104)
2.95 (261)

SD
mmol/L (mg/dL)
0.03 (3)
0.04 (4)
0.02 (2)
0.05 (4)

CV
%
2.0
1.6
1.9
1.8

* repeatability = within-run precision
** intermediate precision = total precision / between run precision / between day precision

Method comparison
Triglycerides values for human serum and plasma samples obtained on a Roche/Hitachi cobas c 501
analyzer (y) were compared with those determined using the same reagent on a Roche/Hitachi 917 analyzer
(x).
Sample size (n) = 71
Passing/Bablok14
Linear regression
y = 1.015x - 0.005 mmol/L
y = 1.001x + 0.018 mmol/L
r = 0.999
τ = 0.976
The sample concentrations were between 0.560 and 9.13 mmol/L (49.6 and 808 mg/dL).

References
1. Greiling H, Gressner AM, eds. Lehrbuch der Klinischen Chemie und Pathobiochemie, 3rd ed.
Stuttgart/New York: Schattauer, 1995.
2. Eggstein M, Kreutz F. Klin Wschr 1966;44:262-267.
3. Bucolo G, David H. Clin Chem 1973;19:476.
4. Wahlefeld AW, Bergmeyer HU, eds. Methods of Enzymatic Analysis. 2nd English ed. New York, NY:
Academic Press Inc, 1974:1831.
5. Trinder P. Ann Clin Biochem 1969;6:24.
6. Siedel J et al. AACC Meeting Abstract 34. Clin Chem 1993;39: 1127.
7. Tietz NW, ed. Clinical Guide to Laboratory Tests, 3rd ed. Philadelphia, Pa: WB Saunders
Company,1995:610-611.
8. Glick MR, Ryder KW, Jackson SA. Graphical Comparisons of Interferences in Clinical Chemistry
Instrumentation. Clin Chem 1986;32:470-475.
9. Shephard MDS, Whiting MJ. Falsely Low Estimation of Triglycerides in Lipemic Plasma by the
Enzymatic Triglyceride Method with Modified Trinder's Chromogen. Clin Chem 1990; Vol 36, No.2,
325-329.
10. Breuer J. Report on the Symposium “Drug effects in Clinical Chemistry Methods”. Eur J Clin Chem
Clin Biochem 1996;34:385-386.
11. Sonntag O, Scholer A. Drug interference in clinical chemistry: recommendation of drugs and their
concentrations to be used in drug interference studies. Ann Clin Biochem 2001;38:376-385.
12. Stein EA, Myers GL. National Cholesterol Education Program Recommendations for Triglycerides
Measurement: Executive Summary. Clin Chem 1995;41:1421-1426.
13. Study Group, European Atherosclerosis Society. Strategies for the prevention of coronary heart disease:
A policy statement of the European Atherosclerosis Society. European Heart Journal 1987;8:77.
14. Passing H, Bablok W et al. A General Regression Procedure for Method Transformation. J Clin Chem
Clin Biochem 1988;26:783-790.

7 of 8 pages

Laboratory Name
Test Name: Triglycerides

Alternative method
_______________________________________________________________________________________
_______________________________________________________________________________________

Source document
Reagent Name: TRIGL
Package Insert Version: 2010-03, V6 English

8 of 8 pages

File Type	application/pdf
File Title	SOPs for ATSDR
Author	RRej
File Modified	2014-10-22
File Created	2011-12-22