Cortisol

ADVIA Centaur System

Principle of the Test

The ADVIA Centaur Cortisol assay is a competitive immunoassay using direct chemiluminescent
technology. Cortisol in the patient sample competes with acridinium ester-labeled cortisol in the Lite
Reagent for binding to polyclonal rabbit anti-cortisol antibody in the Solid Phase. The polyclonal rabbit
anti-cortisol antibody is bound to monoclonal mouse anti-rabbit antibody, which is covalently coupled to
paramagnetic particles in the Solid Phase.
The system automatically performs the following steps:

dispenses 20 L of sample into a cuvette

dispenses 50 L of Lite Reagent and 250 L of Solid Phase and incubates for 5.0 minutes at 37C

separates, aspirates, and washes the cuvettes with reagent water

dispenses 300 L each of Acid Reagent and Base Reagent to initiate the chemiluminescent reaction

reports results according to the selected option, as described in the system operating instructions or in
the online help system

An inverse relationship exists between the amount of cortisol present in the patient sample and the
amount of relative light units (RLUs) detected by the system.

Clinical Application and Usefulness

For in vitro diagnostic use in the quantitative determination of cortisol in serum using the ADVIA
Centaur System.

Specimen Collection and Handling

Specimen Collection
BIOHAZARD

All products or objects that come in contact with human or animal body fluids should
be handled, before and after cleaning, as if capable of transmitting infectious diseases.
Wear facial protection, gloves, and protective clothing.

Serum is the recommended sample type for this assay.

This assay requires 20 L of sample for a single determination. Additional volume is required for
onboard dilutions.

Allow serum samples to clot adequately before centrifugation.

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Samples are free of fibrin or other particulate matter.

Samples are free of bubbles.

Specimen Storage and Stability

Tightly cover and refrigerate specimens at 2 to 8C if the assay is not completed within 8 hours.

Freeze samples at or below -20C if the sample is not assayed within 48 hours.

Freeze samples only once and mix thoroughly after thawing.

Reagents
Storage and Stability

Store the reagents upright at 28 C.

Primary reagents stable until the expiration date on the pack label, or for 10 days onboard the system.

Diluent stable until the expiration date on the pack label, or for 28 consecutive days after accessing
the ancillary reagent pack.

CAUTION:

Discard the primary reagent packs at the end of the onboard stability interval.

Do not use reagents beyond the expiration date.

Ingredients
Reagent ingredients for the ADVIA Centaur Cortisol assay are as follows:
Reagent

Volume

Ingredients

Lite Reagent

2.5 mL/ReadyPack

cortisol (~1.7 ng/mL) labeled with acridinium ester in

buffered saline with sodium salicylate (~50 mg/
mL), sodium azide (0.1%) and preservatives

Solid Phase

12.5 mL/ReadyPack

rabbit anti-cortisol antibody (~1.1 g/mL) bound to

monoclonal mouse anti-rabbit IgG antibody
(~56 g/mL) covalently coupled to paramagnetic
particles in buffered saline with sodium azide (0.1%)
and preservatives

Multi-Diluent 3

5.0 mL/ReadyPack

human plasma with sodium azide (0.1%)

WARNING: Sodium azide can react with copper and lead plumbing to form explosive metal azides. On

disposal, flush reagents with a large volume of water to prevent the buildup of azides, if disposal into a
drain is in compliance with federal, state, and local requirements.

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BIOHAZARD

All products or objects that come in contact with human or animal body fluids should
be handled, before and after cleaning, as if capable of transmitting infectious diseases.
Wear facial protection, gloves, and protective clothing.

Reagents Special Preparation

No special preparation of reagents is required.

Calibration
For detailed procedural information about scheduling a calibration, refer to the ADVIA Centaur
Reference Manual or to the online help system.

Two-point Calibration Interval

Use Calibrator E to perform two-point calibrations.
Perform a two-point calibration every 28 days. Additionally, calibrate when the following conditions
occur:

the lot numbers of the primary reagent packs have changed

system components are repaired or replaced

quality control results do not fall within established ranges

Defining Calibrator Values for Two-point Calibration

1. At the workspace, select Calibration.
2. Select Calibrator Definition.
3. Select Scan Data.
4. Scan the barcodes on the Calibrator Assigned Value Card.
5. Define the LIS field.
6. Review the values on the Calibrator Assigned Value card to ensure that they are correct. Scan again,
if required.
7. Select Save.

Master Curve
Use the barcode reader to enter the Master Curve values from the Master Curve card onto the system.
Ensure that the lot number on the Master Curve matches the lot number of the ReadyPack.

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Defining the Master Curve Using the Barcode Scanner

1. At the workspace, select Calibration.
2. Select Master Curve Definition.
3. At the Calibration-Master Curve Definition window, select Scan Data.
4. Scan the barcodes on the Cortisol Master Curve Card.
5. Select Save.

Quality Control (QC)

BioRad Immunoassay Plus controls levels 1 and 3.
Each control is reconstituted with 5.0 mL of Type 1 de-ionized water.
The reconstituted controls are stable for 7 days when stored at 2-8 C.
See posted QC chart for acceptability limits.

QC Frequency
Analyze at least two levels of quality control material on each day that samples are analyzed.
Analyze all levels of quality control material each time a two-point calibration is performed.

Troubleshooting Out-of-Range QC Values

A QC run is acceptable when all values fall within the expected ranges.
If the Cortisol QC results do not fall within the defined ranges then the run is rejected, and you must take
the following corrective action:

review these instructions to ensure that the assay was performed according to the procedures
recommended by Bayer Diagnostics

verify that the materials are not expired

verify that required maintenance was performed

if necessary, recalibrate the system and then rerun the quality control samples or contact
Bayer Diagnostics for more assistance

Instrument Operation and System Description

The ADVIA Centaur system is an automated, random access, direct chemiluminescent immunoassay
analyzer that offers no-pause reloading of reagents, samples, and supplies. Comprehensive assay groups
are available.
When the sample start button is pressed, the barcode labels on the sample cups are read, sample is
aspirated, reagent is dispensed, and the assay process begins. Paramagnetic particles are magnetically

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separated in the cuvette incubation ring. The addition of hydroxyl groups to complete the flash reaction is
accomplished by the addition of Acid and Base. The chemiluminescent reaction occurs in the
luminometer. The PMT measures the chemical light reaction that takes place.
Refer to Section 4 in the ADVIA Centaur Reference Manual for detailed procedures that describe how to schedule
samples and manage the worklist.

A. System Start Button

There is one (1) main system operation button on the ADVIA Centaur, the Start button. Pressing this
button performs the following actions:

Homes the subsystems.

Starts specimen sampling.

If the Start button is pressed while the system is processing samples, it stops sampling additional
specimens, however it continues to process the specimens in the incubation ring.

B. Start-up
1.

Put the samples racks on the sample entry queue.

2.

Press the Start button.

C. Verify Supplies:
While the system is running, you can manage the following supplies without interrupting the run.

Reagent water supply

Liquid waste container

Acid and Base reagents

Cuvette waste bin

Sample tip waste bin and tip tray waste area

Cuvette supply

Sample tip supply

Ancillary reagent packs

Primary reagent packs

D. Scheduling a Run/Entering a Worklist

You can enter a worklist by different methods:

Automated Worklist
You can send the worklist to the ADVIA Centaur from a Lab Information System (LIS). This is done in a
unidirectional manner (Dynamic Download) or in a bi-directional manner (Host Query).

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Manual (Operator-initiated) Worklist

Scheduling Calibrators
1.

At the workspace, select Worklist and then select Schedule.

2.

Select the Calibrator box.

3.

Select the test you want to schedule for calibration.

4.

The reagent lot that is on the system and any defined calibrators are displayed. Select the
appropriate reagent and calibrator lot combination.

5.

Select Save.

6.

Ensure that the lot numbers of reagent and calibrator are available for system use.

Scheduling Controls
1.

At the workspace, select Worklist and then select Schedule.

2.

Select the Control box.

3.

Select each test you would like to schedule.

4.

Select each control level and lot number you need to run.

5.

Select Save.

Scheduling Patient Samples

1.

At the workspace, select Worklist and then select Schedule.

2.

Select the Patient box.

3.

Enter a patient sample identification number (SID).

4.

Press the Enter key.

5.

Select each test, profile, dilution, and/or replicates needed.

6.

Select Save.

For detailed operating procedural information, refer to the ADVIA Centaur Reference Manual or to the
online help system.

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E. Loading Sample Racks

BIOHAZARD

All products or objects that come in contact with human or animal body fluids should
be handled, before and after cleaning, as if capable of transmitting infectious diseases.
Wear facial protection, gloves, and protective clothing.
CAUTION: Do not load more than one size of sample container in each rack. The rack indicator must be
positioned at the correct setting for the size of sample container.

1. Gently mix the calibrators and controls before dispensing into the sample cups.
2. Load the sample cups containing the calibrators, controls, or patient specimen into any Centaur
rack.
3. Load racks onto the sample entry queue.
4. Press the Start button.

F. Loading Ancillary Reagents

Place the ancillary reagent pack on the ancillary entry queue until the green light comes on. The ADVIA
Centaur moves the pack into the ancillary queue.

G. Loading Primary Reagents

CAUTION: Mix all primary reagent packs by hand before loading them onto the system. Visually inspect
the bottom of the reagent pack to ensure that all particles are dispersed and resuspended. For detailed
information about preparing reagents for use, refer to Appendix C, Handling Reagents, in your ADVIA
Centaur Assay Manual.
1.

Gently mix the Cortisol ReadyPack before placing it on the reagent shelf for the first time.

2.

Load the Cortisol ReadyPack by matching the color on the pack to the color-coded reagent shelf.

3.

Close the reagent compartment door.

H. Loading STAT Samples in the STAT Entry Queue

1.

Press the Start button, if the green Start light is not lit.

2.

Place the STAT sample tube or sample cup in the rack.

3.

Load the rack in the STAT entry queue until the green STAT light comes on, indicating the
system has accepted the rack.

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Performance Characteristics
Precision
Five samples were assayed 6 times, in each of 24 runs, on 6 systems, (n = 144 for each sample),
over a period of 2 days. The following results were obtained:
Mean (g/dL)
3.88
5.63
14.17
27.53
37.15

Mean (nmol/L)
107.05
155.33
390.95
759.55
1024.97

Within-run % CV
3.69
3.09
2.89
3.82
2.98

Run-to-run % CV
5.45
3.83
3.07
1.86
3.99

Total % CV
6.58
4.92
4.22
4.25
4.98

Standardization
The ADVIA Centaur Cortisol assay is standardized using internal standards manufactured
analytically which are traceable to gas chromatography-mass spectroscopy (GCMS). The
following equation describes the relationship between the cortisol standards and GCMS
analysis throughout the range of the assay.
ADVIA Centaur Cortisol = 0.99 (GCMS) + 0.75 g/dL, r = 0.99
Assigned values for calibrators and ranges of Ligand Plus controls are traceable to this
standardization.

Specificity
The Centaur Cortisol assay is highly specific for cortisol.
Cross-reactivity by structurally related compounds and pharmaceuticals was determined by spiking
each compound into separate human serum samples to a final level of 1000 g/dL
(27,590 nmol/L), unless otherwise noted.
% cross-reactivity = (cortisol in spiked sample, g/dL cortisol in unspiked sample, g/dL) x 100
(concentration of compound added, g/dL)
Compound

Aldosterone
Allotetrahydrocortisol
Andrastenedione
Corticosterone

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Concentration of
Compound tested

Updated % CrossReactivity

1000 g/dL
100 g/dL
1000 g/dL
1000 g/dL

0.3
6.5
ND
5.3

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Cortisone
-cortol
-cortolone
-cortol
-cortolone
Dehydrocorticosterone
11-deoxycorticosterone
11-deoxycortisol
21-deoxycortisol
20-dihydrocortisol
20-dihydrocortisol
20-dihydrocortisone
20-dihydrocortisone
11-hydroxyandrosterone
6-hydroxycortisol
11-hydroxyetiocholanolone
11-hydroxyprogesterone
17-hydroxyprogesterone
17-hydroxypregnanolone
11-keto-androsterone
11-keto-etiocholanolone
Pregnanetriol
Pregnenolone
Progesterone
Spironolactone
Testosterone
Tetrahydrocortisol
Tetrahydrocortisone
Tetrahydro-11-deoxycortisol
Prednisolone
6-methyl-prednisolone
Dexamethasone
Prednisone
Canrenone

100 g/dL
1000 g/dL
1000 g/dL
1000 g/dL
1000 g/dL
1000 g/dL
1000 g/dL
100 g/dL
100 g/dL
1000 g/dL
1000 g/dL
1000 g/dL
1000 g/dL
1000 g/dL
1000 g/dL
1000 g/dL
1000 g/dL
1000 g/dL
1000 g/dL
1000 g/dL
1000 g/dL
1000 g/dL
1000 g/dL
1000 g/dL
1000 g/dL
1000 g/dL
1000 g/dL
1000 g/dL
1000 g/dL
50 g/dL
100 g/dL
1000 g/dL
100 g/dL
1000 g/dL

31.1
ND
ND
ND
ND
1.9
1.8
23.3
8.1
0.5
1.3
0.2
ND
ND
6.8
ND
1.0
1.2
ND
ND
1.8
ND
0.5
ND
ND
0.2
ND
0.3
ND
109
26.2
0.2
34.0
0.3

Analytical Sensitivity
The ADVIA Centaur Cortisol assay has an analytical sensitivity of 1 g/dL.
Analytical sensitivity is defined as the concentration of cortisol that corresponds
to the RLUs that are two standard deviations less than the mean RLUs of 20 replicate
determinations of the Cortisol zero standard.

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Analytical Measuring range (AMR)

The analytical measuring range of the ADVIA Centaur Cortisol assay is 1g/dL to 75 g/dL.

Reportable Range
The reportable range of the ADVIA Centaur Cortisol assay is 1g/dL to dilute until an answer is
achieved. Values below 1 g/dL are reported as <1 g/dL.

Dilutions

NOTE: serum samples with cortisol levels greater than 75 g/dL (2069 nmol/L) are
programmed to rerun on a 1:2 instrument performed autodilution.

The sample volume required to perform onboard dilutions (1:2) is 100 L.

For automatic dilutions, ensure that ADVIA Centaur Multi-Diluent 3 is on the Centaur.

Ensure that results are mathematically corrected for dilution.

If upon a 1:2 autodilution the result is >150, then perform a manual dilution until an answer is
achieved. See dilution instructions for details.

Reference Interval

79 a.m. serum: 422 g/dL

35 p.m. serum: 317 g/dL

Units for Reporting Results

The system reports serum cortisol results in g/dL (mass units) or nmol/L (SI units). The conversion
formula is 1 g/dL = 27.59 nmol/L. Units are user-defined in the system software.
The system reports direct urine results in g/dL.

Procedure Notes

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Calculations
For detailed information about how the system calculates results, refer to the ADVIA Centaur Reference
Manual or to the online help system.

Disposal
Dispose of hazardous or biologically contaminated materials according to the practices of your
institution. Discard all materials in a safe and acceptable manner, and in compliance with all federal,
state, and local requirements.

Method Limitations
Circulating cortisol results from patients receiving Prednisolone or Prednisone (which is converted to
Prednisolone in vivo) therapy may be falsely elevated. Caution must be exercised with cortisol
determinations for patients undergoing therapy with these and structurally related synthetic
corticosteroids.
Heterophilic antibodies in human serum can react with reagent immunoglobulins, interfering with in vitro
immunoassays. Patients routinely exposed to animals or to animal serum products can be prone to this
interference and anomalous values may be observed. Additional information may be required for
diagnosis.
Serum specimens that are . . .

Have an insignificant effect on the assay up to . . .

hemolyzed

500 mg/dL of hemoglobin

lipemic

1170 mg/dL of triglycerides

icteric

20 mg/dL of bilirubin

If there is an interferant (hemolysis, lipemia and icterus) higher than the values listed in the above
table, run the sample undiluted and send the corresponding ETC Code:

TUR Specimen turbid, result may be invalid

ICTRQ Specimen icteric, result may be invalid
HEMRQ Specimen hemolyzed, result may be invalid

For additional information on performance characteristics including cross reactivity and dilution recovery,
see the product information in the ADVIA Centaur Assay Manual.

Equipment and Supplies

ADVIA Centaur Cortisol ReadyPack

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ADVIA Centaur Calibrator E

ADVIA Centaur Multi-Diluent 3

ADVIA Centaur Sample Cups and Caps

ADVIA Centaur Cuvettes

ADVIA Centaur Tips

ADVIA Centaur Cleaning Solution Concentrate

ADVIA Centaur Acid Reagent (0.5% H202, 0.1N HNO3)

ADVIA Centaur Base Reagent (0.25N NaOH and surfactant)

Reagent Water

References
1.

Bayer Diagnostics ADVIA Centaur Cortisol product insert, Revision E.

2.

Bayer Diagnostics ADVIA Centaur Reference Manual, Revision D.

3.

Bayer Diagnostics ADVIA Centaur Assay Manual, Revision AT.

4.

National Committee for Clinical Laboratory Standards (NCCLS). Clinical Laboratory Procedure
ManualsThird Edition (GP2-A3), 1996.

Technical Assistance
Bayer Diagnostics Technical Care Center: 1-877-229-3711
Customer Service: 1-800-255-3232
Serial Number: IRL 80600727
Customer Account Number: 619504

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