STANDARD OPERATING PROCEDURE
CORTISOL
1. PURPOSE OF EXAMINATION:
a. To measure Cortisol levels quantitatively in the specimen by Automated
Method.
b. To use Cortisol Measurements as an aid in the diagnosis of hypothalamic,
pituitary or adrenal malfunction.
2. PRINCIPLE OF TEST METHOD:
The VITROS Cortisol assayis performed using the VITROS Cortisol Reagent Pack and
VITROS Immunodiagnostic Products Cortisol Calibrators on the VITROS
Immunodiagnostic System. A competitive immunoassay technique is used, which
depends on a competition between cortisol present in the sample with a horseradish
peroxidase (HRP)-labeled cortisol conjugate for a limited number of binding sites on
a biotinylated antibody(sheep anti-cortisol). The antigen-antibodycomplex is
captured by streptavidin on the wells, unbound materials are removed by washing.
The bound HRP conjugate is measured by a luminescent reaction7. A reagent
containing luminogenic substrates (a luminol derivative and a per acid salt) and an
electron transfer agent, is added to the wells. The HRP in the bound conjugate
catalyzes the oxidation of the luminol derivative, producing light. The electron
transfer agent (as ubstituted acetanilide) increases the level of light produced and
prolongs its emission. The light signals are read by the VITROS System. The amount
of HRP conjugate bound is indirectly proportional to the concentration of cortisol
present.
3. SPECIMEN COLLECTION AND HANDLING:
A. Primary sample/Patient preparation:
Whole blood collected by venipuncture; Vacutainer containing blood up to the
mark, is gently inverted 5 times to ensure proper mixing of specimen with clot
activator.
B. Specimen type required (secondary sample): Serum
Laboratory Medicine & Pathology
Doc No.:LMP/IA 03 Document Name: Cortisol
Issue No.: Effective Date: Next Review Date: 18/09/2018 Review by: Dev Shankar Yadav
17/09/2017 Designation: Technical Manager
Page | 1 of 7
Amendment No.: Amendment Date: Prepared by: Dil Bahadur Saru Approved by: Dr. Gopi Aryal Issued by: Nageshwar Chaudhary
Designation: Section Incharge Designation: Laboratory Director Designation: Quality Manager
STANDARD OPERATING PROCEDURE
C. Other specimens analysed by this method: Plasma, Urine
D. Type of container and additives for
i. Serum: Red capped vacutainer containing Clot activator/SST
Ii. Plasma: Anticoagulant- Heparin, EDTA.
E. Sample separation: Serum, plasma should be separated from cells within 2 hrs of
collection. Blood Samples are allowed to clot completely at room temperature (RT)
for 15 to 30 minutes. Blood samples are centrifuged at 3000 rpm for 10 minutes.
F. Rejection criteria: Unlabeled, wrongly labeled, contaminated specimens;
Serum/plasma exceeding HIL interference limits (refer 5g).
G. Sample stability and storage:
Serum/plasma free from evaporation effects is stable at room temperature
(RT) for 8 hrs, at 2-8°C for 48 hrs, at -20°C for longer storage. Samples may
be thawed only once.
Post test sample storage by the lab: Sample portions at -20°C, for 3 days.
4. SPECIFIC PERFORMANCE CHARACTERISTICS:
a. Assay Range/AMR : 0.20 – 75 μg/dL (5.5 – 2069 nmol/L)
b. Analytical Sensitivity : 0.20μg/dL
c. Calibration and Its Verification :The details of calibration carried out during
validation are available in the validation Report.
i. Acceptable CV for Low Cal : 9.3
ii. Acceptable CV for High Cal : 9.0
iii. Master Curve Data : Critical Precision is 0.995
d. Measurement Imprecision:
i. Measurement repeatability( Within-run/Intra run) : Level 1 is 0.9, Level 2 is
9.1 and Level 3 is 1.5.
ii. Measurement reproducibility (Inter assay) : Level 1 is 8.89, Level 2 is 9.1
and Level 3 is 7.99.
Laboratory Medicine & Pathology
Doc No.:LMP/IA 03 Document Name: Cortisol
Issue No.: Effective Date: Next Review Date: 18/09/2018 Review by: Dev Shankar Yadav
17/09/2017 Designation: Technical Manager
Page | 2 of 7
Amendment No.: Amendment Date: Prepared by: Dil Bahadur Saru Approved by: Dr. Gopi Aryal Issued by: Nageshwar Chaudhary
Designation: Section Incharge Designation: Laboratory Director Designation: Quality Manager
STANDARD OPERATING PROCEDURE
e. Measurement accuracy : 12.6 %
f. Method comparison: For 394 serum samples in the range of 0.49 to 58.76
μg/dL (13.52 to 1621.24 nmol/L), the Relationship between the ADVIA
Centaur Cortisol assay and the ACS: 180 Cortisol assay is described by the
equation:
ADVIA Centaur Cortisol = 1.01 (ACS: 180 Cortisol) + 0.26 μg/dL
Correlation coefficient (r) = 0.99
For 109 direct urine samples in the range of 18.9 to 307.7 μg/24 hours (52.2
to 849.3 nmol/24 hours), the relationship between the ADVIA Centaur
Cortisol assay and the ACS:180 Cortisol assay is described by the equation:
ADVIA Centaur Cortisol = 1.06 (ACS:180 Cortisol) + 0.404 μg/24 hours
Correlation coefficient (r) = 0.99
g. Interferences / Analytical specificity: HIL (hemolysis, icterus & lipemia)
interference.
No significant interference in serum/plasma with Hemolysis (Hemoglobin
upto 500 mg/dL), Icterus (Bilirubin upto 20 mg/dL), Lipaemia (upto 1170
mg/dl Triglycerides).
5. REQUIRED EQUIPMENTS AND REAGENTS:
Equipments: ADVIA Centaur XP systems from Siemens Healthcare Diagnostics,
micropipettes, refrigerators, centrifuges.
Reagents (Refer to pack insert): ADVIA Centaur COR Ready Pack primary reagent
pack. Lite Reagent, Solid Phase.Liquid reagent, stored at 2-8 °C and ready-to-use
Onboard Stability: Until expiry date for sealed reagent packs and 7 days for open
wells
6.CALIBRATION PROCEDURES & metrological traceability: refer advia centaur xp
assay manual.
a. Calibrator: ADVIA CENTAUR Calibrator E
Laboratory Medicine & Pathology
Doc No.:LMP/IA 03 Document Name: Cortisol
Issue No.: Effective Date: Next Review Date: 18/09/2018 Review by: Dev Shankar Yadav
17/09/2017 Designation: Technical Manager
Page | 3 of 7
Amendment No.: Amendment Date: Prepared by: Dil Bahadur Saru Approved by: Dr. Gopi Aryal Issued by: Nageshwar Chaudhary
Designation: Section Incharge Designation: Laboratory Director Designation: Quality Manager
STANDARD OPERATING PROCEDURE
Calibrator traceability: 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 μg/dL, r = 0.99
Assigned values for calibrators and ranges of Ligand Plus controls are traceable to
this standardization.
a. Calibration Scheme: 2 levels of calibrators in triplicate.
b. Calibration Frequency:
i. Every new lot of reagent cartridge.
ii. Every 10 days for any one lot.
iii. As indicated in laboratory quality control procedures.
iv. After major maintenance or service, if indicated by IQC results.
7. PROCEDURAL STEPS:
The VITROS Cortisol assay requires 25 µL sample, calibrator or control for a singleton
determination. This does not take account of the minimum fill volume of the chosen
sample container.
The VITROS Cortisol assay must be calibrated each time a new reagent lot is used,
and subsequently at intervals of 28 days.
The VITROS Cortisol assay may also need to be calibrated after certain service
procedures are performed or if quality control results are consistently outside your
acceptable range.
For detailed instructions on the operation of the System refer to the VITROS
Immunodiagnostic System Operatorís Guide.
Chapters 4-7. In summary:
1. Scan the protocol card to load a new assay protocol onto the System. The assay
button is then displayed on the Sample Programming screen. Scan the lot calibration
Laboratory Medicine & Pathology
Doc No.:LMP/IA 03 Document Name: Cortisol
Issue No.: Effective Date: Next Review Date: 18/09/2018 Review by: Dev Shankar Yadav
17/09/2017 Designation: Technical Manager
Page | 4 of 7
Amendment No.: Amendment Date: Prepared by: Dil Bahadur Saru Approved by: Dr. Gopi Aryal Issued by: Nageshwar Chaudhary
Designation: Section Incharge Designation: Laboratory Director Designation: Quality Manager
STANDARD OPERATING PROCEDURE
card for each new reagent lot to enter lot specific calibration and expiration
information.
2. Open the foil pouch and remove the reagent pack. Load the pack at the auto-load
station, or use the Unload/Load button on the Reagent Management – View by
Reagent screen.
NOTE: Do not use damaged or incompletely sealed product.
3. Load samples into universal sample trays, using adapters where
necessary(samples maybe bar coded if desired). Place a disposable tip adjacent to
each sample and load the trays onto the System. Define sample programs using the
Sample Programming screen. Start the sampling operation, all sample processing
steps will be carried out automatically.
4. Process calibrators in the same manner as samples. Transfer an aliquot of each
calibrator into a sample container (taking account of the minimum fill volume of the
container), which maybe bar coded with the labels provided. Calibration need not be
programmed if bar code labels are used; load the calibratorsin any order, calibration
will be initiated automatically.
8. QUALITY CONTROL PROCEDURES:
a. Internal Quality Control (IQC): IQC material with known Cortisol
concentrations are analyzed on the days of use and data is reviewed.
QC product material:
Serum: LYPHOCHECK Immunoassay Plus Control (IPC), Level I, II and II from
Bio-Rad Laboratories.
Frequency: Any two levels IPC once a day.
Lab mean, SD and CV% is established for the IQC. If the value lies
within the cut off limits as specified by the lab or pack insert for Quality
Control (before establishing the lab mean), the test results are
released. Levey-Jennings control charts are maintained.
b. Proficiency Testing programme:
i. External Quality Assurance Services (EQAS), Bio-Rad Laboratories:
Participation in Monthly Clinical Chemistry Quality Assessment Programme.
Laboratory Medicine & Pathology
Doc No.:LMP/IA 03 Document Name: Cortisol
Issue No.: Effective Date: Next Review Date: 18/09/2018 Review by: Dev Shankar Yadav
17/09/2017 Designation: Technical Manager
Page | 5 of 7
Amendment No.: Amendment Date: Prepared by: Dil Bahadur Saru Approved by: Dr. Gopi Aryal Issued by: Nageshwar Chaudhary
Designation: Section Incharge Designation: Laboratory Director Designation: Quality Manager
STANDARD OPERATING PROCEDURE
SDI and Z score are monitored. SDI < 2.0 and Z score < 2 indicate
acceptable performance.
9. PRINCIPLE OF PROCEDURE FOR CALCULATING RESULTS
Chemiluminometric principle employs the calculation based on the measurement of
light emitted when an electron returns from an excited state to ground state
(Excitation event is by a chemical reaction). The instrument automatically calculates
results from the absorbance and prints the concentration of Cortisol in μg/dL.
To convert from conventional units to S. I. units: μg/dL x 27.59 = nmol/L
10. REPORTABLE INTERVAL OF PATIENT EXAMINATION RESULTS
Reportable range: (4.3 – 1700 nmol/L)
Lowest value reported: <4.3 nmol/L
Reporting of test values above the reportable range (Clinical reportable
range):
Serum : >150 μg/dL
11.BIOLOGICALREFERENCEINTERVALS:
7-9AM 4.3- 22.4
Unit: μg/dL
4-6PM 3.9–16.66
12. ALERT/ CRITICAL VALUES, WHERE APPROPRIATE: NA
13. CLINICAL SIGNIFICANCE:
Increase Decrease
Cushing’s disease; Adrenal adenoma, Addison’s disease; congenital adrenal
carcinoma; Ectopic ACTH syndrome hyperplasia; Hypopitutarism
14. LABORATORY INTERPRETATION: Done, where appropriate in consultation with
Laboratory Medicine & Pathology
Doc No.:LMP/IA 03 Document Name: Cortisol
Issue No.: Effective Date: Next Review Date: 18/09/2018 Review by: Dev Shankar Yadav
17/09/2017 Designation: Technical Manager
Page | 6 of 7
Amendment No.: Amendment Date: Prepared by: Dil Bahadur Saru Approved by: Dr. Gopi Aryal Issued by: Nageshwar Chaudhary
Designation: Section Incharge Designation: Laboratory Director Designation: Quality Manager
STANDARD OPERATING PROCEDURE
clinician.
15. SAFETY PRECAUTIONS: Refer to Advia centaur assay manual for cortisol.
16. POTENTIAL SOURCES OF VARIABILITY:
1. Refer to Pack insert for interferences from various substances and drugs
2. Interferences from pre analytical, analytical and post analytical phases
3. Diurnal variation
4. Pregnancy
17. REFERENCES:
Vitros 5600 Cortisol reagent cartridge & calibrator pack insert sheets
Operator’s guide – vitros 5600
TEITZ Textbook of Clinical Chemistry- Burtis. C.A. Ashwood E. R. 3rd edition.
TEITZ Textbook of Clinical Chemistry & Molecular Diagnostics- Burtis. C.A.
Ashwood E. R. 4th editions.
Clinical Guide to Laboratory Tests, 3rd edition- Norbert W. Teitz, 4th edition-
Alan H.B WY.
A manual of laboratory and diagnostic tests – Francis Fischbach 6th edition.
Medical Laboratory technology 6th edition by Ramnik Sood.
Laboratory Medicine & Pathology
Doc No.:LMP/IA 03 Document Name: Cortisol
Issue No.: Effective Date: Next Review Date: 18/09/2018 Review by: Dev Shankar Yadav
17/09/2017 Designation: Technical Manager
Page | 7 of 7
Amendment No.: Amendment Date: Prepared by: Dil Bahadur Saru Approved by: Dr. Gopi Aryal Issued by: Nageshwar Chaudhary
Designation: Section Incharge Designation: Laboratory Director Designation: Quality Manager
