INSTRUCTIONS FOR USE

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1 INSTRUCTIONS FOR USE VITROS Immunodiagnostic Products Cortisol Reagent Pack VITROS Immunodiagnostic Products Cortisol Calibrators Cort Rx ONLY Intended Use For in vitro diagnostic use only. VITROS Immunodiagnostic Products Cortisol Reagent Pack For the quantitative measurement of cortisol in human serum, plasma (heparin or EDTA) or urine using the VITROS ECi/ ECiQ Immunodiagnostic Systems, the VITROS 3600 Immunodiagnostic System and the VITROS 5600 Integrated System. VITROS Immunodiagnostic Products Cortisol Calibrators For use in the calibration of the VITROS Immunodiagnostic Systems, the VITROS 3600 Immunodiagnostic System and the VITROS 5600 Integrated System for the quantitative measurement of cortisol in human serum, plasma (heparin or EDTA) or urine. Summary and Explanation of the Test Corticosteroids are synthesized from cholesterol in the adrenal cortex, and cortisol is the main corticosteroid produced and secreted by humans. 1 Physiological actions of cortisol include regulation of carbohydrate metabolism, electrolyte balance, water distribution and immunosuppressant and anti-inflammatory activity. 2 Cortisol secretion is dependent on the integrity of the hypothalamic-pituitary-adrenal (HPA) axis and the steroid exerts a negative feedback on its own synthesis through this axis. 3 Cortisol measurement is thus an important parameter in the investigation of apparent HPA dysfunction. Cortisol circulates largely in protein-bound forms, the majority being attached to corticosteroid binding globulin. 4 The halflife of cortisol in the circulation is about 80 minutes, 5 with approximately 1% excreted unchanged in the urine. This excreted fraction is called urinary free cortisol and if renal function is normal, will reflect the level of circulating non-protein bound cortisol. Most immunological methods employed for the determination of urinary free cortisol omit chromatographic steps thereby co measuring cortisol metabolites. After metabolic breakdown, mainly in the liver, cortisol is excreted into the urine as dihydro and tetrahydro-derivatives conjugated to glucuronic acid. The circulating cortisol concentration is normally subject to a circadian rhythm, with the maximum level being reached at 8 9 a.m. and the minimum around midnight. 5, 6 Concentrations are usually elevated in pregnancy and in patients receiving high dose estrogen therapy. 4 Anomalous cortisol concentrations can result from stimuli such as trauma, fear, fever, shock, hypoglycemia and depression. In pathological states of the HPA axis, elevated or depressed values of cortisol may be found. Adrenal tumors and pituitary or ectopic adrenocorticotrophic hormone (ACTH)-producing tumors are frequently associated with cortisol over-production (Cushing s syndrome), while adrenal insufficiency results in cortisol under-production (Addison s disease). Principles of the Procedure The VITROS Cortisol test is performed using the VITROS Cortisol Reagent Pack and the VITROS Cortisol Calibrators on the VITROS Immunodiagnostic Systems, the VITROS 3600 Immunodiagnostic System and the VITROS 5600 Integrated System using Intellicheck Technology. 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-antibody complex is captured by streptavidin on the wells. Unbound materials are removed by washing. The bound HRP conjugate is measured by a luminescent reaction. 7 A reagent containing luminogenic substrates (a luminol derivative and a peracid 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 (a substituted acetanilide) increases the level of light produced and prolongs its emission. The light signals are read by the system. The amount of HRP conjugate bound is indirectly proportional to the concentration of cortisol present. Version 8.0 Pub. No. GEM1401_WW_EN 1 of 16

2 Cort INSTRUCTIONS FOR USE Warnings and Precautions Test Type System * Incubation Time Time to first result Test Temperature Competitive immunoassay, 5600, 3600 * Not all products and systems are available in all countries. Reaction Sample Volume 30 minutes 38 minutes 37 C 25 μl Reaction Scheme Warnings and Precautions WARNING: Potentially Infectious Material Human blood products provided as components of the VITROS Cortisol Reagent Pack and the VITROS Cortisol Calibrators have been obtained from donors who were tested individually and who were found to be negative for hepatitis B surface antigen, and for antibodies to human immunodeficiency virus (HIV 1+2) and hepatitis C virus (HCV), using approved methods (enzyme immunoassays). Treat as if capable of transmitting infection. Use caution when handling material of human origin. Consider all samples potentially infectious. No test method can offer complete assurance that hepatitis B virus, HCV, HIV 1+2 or other infectious agents are absent. Handle, use, store and dispose of solid and liquid waste from samples and test components, in accordance with procedures defined by appropriate national biohazard safety guideline or regulation (e.g. CLSI document M29). 8 WARNING: Contains Kathon or ProClin 200 (CAS ) 9 The VITROS Cortisol Reagent Pack and VITROS Cortisol Calibrators contain 2% and 1% Kathon or ProClin 200 respectively. H317: May cause an allergic skin reaction. P280: Wear protective gloves/protective clothing/eye protection/face protection. P302 + P352: IF ON SKIN: Wash with plenty of soap and water. P333 + P313: If skin irritation or rash occurs: Get medical advice/attention. P363: Wash contaminated clothing before reuse. Refer to for the Safety Data Sheets and for OCD contact information. WARNING Reagents Reagent Pack Contents 1 reagent pack containing: 2 of 16 Pub. No. GEM1401_WW_EN Version 8.0

3 INSTRUCTIONS FOR USE Reagents Cort 100 coated wells (streptavidin, bacterial; binds 3 ng biotin/well) 9.1 ml conjugate reagent (HRP-cortisol, 100 ng/ml) in buffer with human plasma, bovine serum albumin, bovine gamma globulin and antimicrobial agent 9.1 ml biotinylated antibody reagent (biotin-sheep polyclonal anti-cortisol; binds nmol cortisol/ml) in buffer with bovine serum albumin and antimicrobial agent Reagent Pack Handling The reagent pack is supplied ready for use. The reagent pack contains homogeneous liquid reagents that do not require shaking or mixing prior to loading onto the system. Handle the reagent pack with care. Avoid the following: allowing condensation to form on the pack causing reagents to foam agitation of the pack Reagent Pack Storage and Preparation Reagent Storage Condition Stability Unopened Refrigerated 2 8 C (36 46 F) expiration date Opened 3600/5600 (Lot 1738 and above): On system 3600/5600 (Lot 1730 and below): On system : On system Opened 3600/5600 (Lot 1738 and above): Refrigerated 3600/5600 (Lot 1730 and below): Refrigerated : Refrigerated System turned on System turned on System turned on 7 weeks 6 weeks 8 weeks 2 8 C (36 46 F) 7 weeks 2 8 C (36 46 F) 6 weeks 2 8 C (36 46 F) 8 weeks The VITROS Cortisol Reagent Pack is suitable for use until the expiration date on the carton when stored and handled as specified. Do not use beyond the expiration date. Do not freeze unopened reagent packs. Load reagent packs directly from refrigerated storage to minimize condensation. Store opened refrigerated reagent packs in a sealed reagent pack storage box that contains dry desiccant. Calibrator Contents 3 sets of VITROS Cortisol Calibrators 1, 2 and 3 (freeze-dried cortisol in human plasma with antimicrobial agent, reconstitution volume 1 ml); nominal values 0; 125 and 750 nmol/l (0; 4.53 and 27.2 μg/dl) Lot calibration card Protocol card 24 calibrator bar code labels (8 for each calibrator) Calibrator Handling Use only with reagent packs of the same lot number. Mix thoroughly by inversion and bring to C (59 86 F) before use. Each pack contains sufficient for a minimum of 6 determinations of each calibrator. Handle calibrators in stoppered containers to avoid contamination and evaporation. To avoid evaporation, limit the amount of time calibrators are on the system. Refer to the operating instructions for your system. Return to 2 8 C (36 46 F) as soon as possible after use, or load only sufficient for a single determination. Version 8.0 Pub. No. GEM1401_WW_EN 3 of 16

4 Cort INSTRUCTIONS FOR USE Specimen Collection, Preparation and Storage Calibrator Storage and Preparation Calibrator Storage Condition Stability Unopened Refrigerated 2 8 C (36 46 F) expiration date Openedreconstituted Openedreconstituted Refrigerated 2 8 C (36 46 F) 13 weeks Frozen -20 C ( -4 F) 13 weeks VITROS Cortisol Calibrators are supplied freeze-dried. VITROS Cortisol Calibrators are suitable for use until the expiration date on the carton when stored and handled as specified. Do not use beyond the expiration date. Reconstitute with 1 ml distilled water. Opened, reconstituted calibrators may be stored frozen (with no more than 1 freeze-thaw cycle). The VITROS Cortisol test uses 25 μl of calibrator for each determination. Transfer an aliquot of each calibrator into a sample container (taking account of the minimum fill volume of the container), which may be bar coded with the labels provided. For details on minimum fill volume of sample cups or containers, refer to the operating instructions for your system. Specimen Collection, Preparation and Storage Patient Preparation No special patient preparation is necessary. Specimens Recommended Serum Heparin plasma EDTA plasma 24 hour urine Note: Urine specimens must be extracted with dichloromethane prior to processing (see Extraction Pretreatment for Urine Samples). Do not add preservative to urine specimens. Specimens Not Recommended Do not use turbid specimens. Turbidity in specimens may affect test results. Do not use hemolysed samples as hemolysis may affect test results. Special Precautions IMPORTANT: Certain collection devices have been reported to affect other analytes and tests. 10 Owing to the variety of specimen collection devices available, Ortho Clinical Diagnostics is unable to provide a definitive statement on the performance of its products with these devices. Confirm that your collection devices are compatible with this test. Specimen Collection and Preparation Collect specimens using standard procedures Thoroughly mix samples by inversion and bring to C (59 86 F) before use. The VITROS Cortisol test uses 25 μl of sample for each determination. This does not take account of the minimum fill volume of the chosen sample container. For details on minimum fill volume of sample cups or containers, refer to the operating instructions for your system. Handling and Storage Conditions Handle samples in stoppered containers to avoid contamination and evaporation. The amount of time samples are on the system prior to analysis should be limited to avoid evaporation. Refer to the operating instructions for your system. Return to 2 8 C (36 46 F) as soon as possible after use, or load sufficient volume for a single determination. Serum and plasma samples may be stored for up to 5 days at 2 8 C (36 46 F) or 4 weeks at -20 C (-4 F). Urine samples may be stored for up to 5 days at 2 8 C (36 46 F) or 4 weeks at -20 C (-4 F). Avoid repeated freeze thaw cycles. 4 of 16 Pub. No. GEM1401_WW_EN Version 8.0

5 INSTRUCTIONS FOR USE Testing Procedure Cort Testing Procedure Materials Provided VITROS Immunodiagnostic Products Cortisol Reagent Pack VITROS Immunodiagnostic Products Cortisol Calibrators Materials Required but Not Provided VITROS Immunodiagnostic Products Signal Reagent VITROS Immunodiagnostic Products Universal Wash Reagent VITROS Immunodiagnostic Products High Sample Diluent B Quality control materials such as VITROS Immunodiagnostic Products Metabolism Controls VITROS Immunodiagnostic Products Reagent Pack Storage Box (optional) with desiccant Dichloromethane (analytical grade, for the extraction of urine samples) Calibrated pipette, distilled water and sample containers for reconstitution of VITROS Cortisol Calibrators Calibrated glass pipettes capable of accurately delivering volumes of 0.15 ml, 0.25 ml, 0.75 ml, and 1.25 ml for the extraction of urine samples Test tubes Operating Instructions Check the inventory regularly to aid the management of reagents and ensure that sufficient VITROS Signal Reagent, VITROS Universal Wash Reagent and calibrated reagent lots are available for the work planned. When performing panels of tests on a single sample, ensure that the sample volume is sufficient for the tests ordered. For detailed information refer to the operating instructions for your system. Note: Do not use visibly damaged product. Extraction Pretreatment for Urine Samples For handling, use, storage and waste disposal of dichloromethane, refer to the instructions for use provided by the manufacturer. 1. Record the urine specimen volume in liters. 2. Pipette 250 μl of urine specimen into a clean glass test tube. 3. Add 1.25 ml chilled dichloromethane. 4. Vortex intermittently for 20 seconds, then place the tube in an ice bath for a minimum of 5 minutes to separate. 5. Remove the top layer and discard. 6. Using a calibrated glass pipette, pipette 750 μl of the lower, organic layer into a clean glass test tube. Use proper pipetting techniques to avoid any spilling of the organic solvents and to ensure accurate volumetric transfer of the cortisol containing layer. 7. Evaporate off solvent by passing air or nitrogen over the liquid. 8. Re-suspend with 150 μl VITROS High Sample Diluent B. 9. Transfer the entire volume to a microsample cup and test on the system. No correction factor for dilution of the specimen is required in interpreting the results. 10. Data must be corrected for 24-hour volume. Refer to Correction of Extracted Urine Cortisol Results for the calculation. Sample Dilution Serum, plasma (heparin or EDTA) samples with concentrations greater than the measuring range may be automatically diluted on the system up to 2-fold (1 part sample with 1 part diluent) by the VITROS Immunodiagnostic and VITROS Integrated Systems with the VITROS High Sample Diluent B Reagent Pack prior to test. Refer to the VITROS High Sample Diluent B Reagent Pack instructions for use. The dilution of urine samples in the VITROS Cortisol test is not supported. Default Test Name The default test name which will appear on patient reports is Cortisol. The default short name that will appear on the test selection menus and laboratory reports is Cort. These defaults may be reconfigured, if required. For detailed information refer to the operating instructions for your system. Version 8.0 Pub. No. GEM1401_WW_EN 5 of 16

6 Cort INSTRUCTIONS FOR USE Calibration Calibration Calibration Procedure Calibration is lot specific; reagent packs and calibrators are linked by lot number. Reagent packs from the same lot may use the same calibration. A Master Calibration (a dose response curve covering the full calibration range) is established for each new reagent lot. Concentrations for the linked lot of calibrators are determined from the Master Calibration. Ensure that the Master Calibration for each new reagent lot is available on your system. Process calibrators in the same manner as samples. Calibration need not be programmed if bar code labels are used; load the calibrators in any order, calibration will be initiated automatically. When the calibrators are processed the signal expected for each calibrator is compared against the actual signal obtained. The Master Calibration is then rescaled to reflect the differences between the actual and expected signals. The validity of this calibration curve is assessed against a range of quality parameters, and if acceptable, it is stored for use with any reagent pack of that lot. The quality of calibration cannot be completely described by a single parameter. The calibration report should be used in conjunction with acceptable control values to determine the validity of the calibration. Recalibration is required after a pre-determined calibration interval, or when a different reagent lot is loaded. Calibration results are assessed against a range of quality parameters. Failure to meet any of the defined quality parameter ranges will be coded in the calibration report. For actions to be taken following a failed calibration refer to the operating instructions for your system. Refer to the operating instructions for your system for detailed instructions on the calibration process. When to Calibrate Calibrate when the reagent pack and calibrator lot changes. Calibrate every 28 days. After specified service procedures have been performed. If quality control results are consistently outside of your acceptable range. For additional information on when to calibrate, refer to the operating instructions for your system. Traceability of Calibration Calibration of the VITROS Cortisol test is traceable to in-house reference calibrators which have been value assigned to correlate to samples measured by gas-chromatography/mass spectrometry (GCMS). Calibration Model A modified four parameter logistic curve fit function is used to construct the Master Calibration. The calibration process rescales the Master Calibration to establish a valid stored curve for the VITROS Immunodiagnostic and VITROS Integrated Systems. Measuring (Reportable) Range System Measuring (Reportable) Range 4.39 * 1700 nmol/l ( μg/dl) * lower limit of measuring range reported by the system software is based on the Limit of Quantitation. The lower limit reported by the system can be reconfigured if desired. For details on how to reconfigure the lower limit refer to the operating instructions for your system. Quality Control Quality Control Material Selection VITROS Metabolism Controls are recommended for use with the VITROS Immunodiagnostic and VITROS Integrated Systems. The VITROS Metabolism Controls contain 3 levels of cortisol (low, medium and high). These controls can also be processed using the urine extraction pretreatment to verify performance of the urine extraction protocol. The performance of other commercial control fluids should be evaluated for compatibility with this test before they are used for quality control. Control materials may show a difference when compared with other cortisol methods if they contain high concentrations of preservatives, stabilizers, or other nonphysiological additives, or otherwise depart from a true human sample matrix. Appropriate quality control value ranges must be established for all quality control materials used with the VITROS Cortisol test. 6 of 16 Pub. No. GEM1401_WW_EN Version 8.0

7 INSTRUCTIONS FOR USE Results Cort Results Quality Control Procedure Recommendations Good laboratory practice requires that controls be processed to verify the performance of the test. Choose control levels that check the clinically relevant concentrations. To verify system performance, analyze control materials: After calibration According to local regulations or at least once per day that the test is being performed After specified service procedures are performed If quality control procedures within your laboratory require more frequent use of controls, follow those procedures. Analyze quality control materials in the same manner as patient specimens. If control results fall outside your acceptable range, investigate the cause before deciding whether to report patient results. Refer to published guidelines for general quality control recommendations. 14 For more detailed information, refer to the operating instructions for your system. Quality Control Material Preparation and Storage Refer to the manufacturer s product literature for preparation, storage, and stability information. Results are automatically calculated by the VITROS Immunodiagnostic and VITROS Integrated Systems. Reporting Units and Unit Conversion Analyte results are quoted in units of nmol/l or μg/dl. To configure the units, refer to the operating instructions for your system. Conventional Alternate nmol/l (μg/dl 27.59) μg/dl (nmol/l ) Correction of Extracted Urine Cortisol Results Determine the results as follows: nmol cortisol/24 hours = nmol cortisol/l 24-hour urine volume (L) µg cortisol/24 hours = µg cortisol/dl hour urine volume (L) Limitations of the Procedure Known Interferences The VITROS Cortisol test was evaluated for interference consistent with CLSI document EP7. 15 Commonly encountered substances were tested on 2 lots of reagents. Of the compounds tested, hemoglobin may interfere with the VITROS Cortisol test. Hemoglobin, when tested, caused the bias shown at the concentrations indicated. Refer to Specificity for a list of other compounds tested that did not show interference. Interferent Interferent Concentration Units = nmol/l Units = μg/dl Analyte Conc * Bias ** Analyte Conc * Bias ** Hemoglobin mmol/l 100 mg/dl Hemoglobin mmol/l 500 mg/dl * Average test concentration of replicate determinations using 2 different lots of reagent. ** Estimate of the average difference observed. Note: Other Limitations These results are representative. The degree of interference at concentrations other than those listed might not be predictable from these results. Other interfering substances may be encountered in the patient population. The results from this or any other diagnostic test should be used and interpreted only in the context of the overall clinical picture. Biotin levels in serum remain elevated for up to 24 hours after oral or intravenous biotin administration. 16 Heterophilic antibodies in serum or plasma samples may cause interference in immunoassays These antibodies may be present in blood samples from individuals regularly exposed to animals or who have been treated with animal serum products. Results which are inconsistent with clinical observations indicate the need for additional testing. Version 8.0 Pub. No. GEM1401_WW_EN 7 of 16

8 Cort INSTRUCTIONS FOR USE Expected Values and Interpretation of Results Materials such as commercially available controls or proficiency panel members containing azide at concentrations greater than or equal to 0.05% have been shown to interfere with this test. Cortisol concentrations can be changed rapidly by strong stimuli and are subject to a daily rhythm so that a single determination yields limited information and should be used with other tests of HPA function (see Summary and Explanation of the Test). Results from patients receiving steroids, especially prednisolone, should be treated with caution due to possible cross reactivity with the sheep anti-cortisol antibody (see Specificity section). Certain drugs and clinical conditions are known to alter cortisol concentrations in vivo. Please consult one of the published summaries for details Expected Values and Interpretation of Results It is recommended that each laboratory establish its own expected values for the population it serves. Reference Interval Serum Sample Collection Time Number of Samples nmol/l Reference Interval μg/dl Before 10:00 am After 5:00 pm Reference intervals (100 samples) were obtained from the sera of apparently healthy patients sampled at the times indicated. Urinary Free Cortisol No. of Samples nmol cortisol/24 hours Reference Interval µg cortisol/24 hours This reference interval was obtained from 24-hour urine samples collected from 42 apparently healthy patients. Interpretation of Results For patient sample values outside your established reference interval, the system may be configured to display a flag LO or HI. Refer to the operating instructions for your system. Performance Characteristics Limit of Detection The Limit of Detection (LoD) for VITROS Cortisol is 2.83 nmol/l (0.10 μg/dl), determined consistent with NCCLS document EP17 22 and with proportions of false positives (α) less than 5% and false negatives (β) less than 1%; based on 1578 determinations, with 214 blank and 1364 low-level samples. The Limit of Blank (LoB) is 0.41 nmol/l (0.01 μg/dl). The Limit of Quantitation (LoQ) is 4.39 nmol/l (0.16 μg/dl) as determined by the lowest concentration at which precision and accuracy design requirements are still met and within the linear range of the test. For the VITROS 3600 Immunodiagnostic System and the VITROS 5600 Integrated System, the LoB, LoD and LoQ were verified consistent with NCCLS document EP The data presented are a representation of the product performance. Limit of Blank, Limit of Detection and Limit of Quantitation LoB * LoD ** LoQ nmol/l μg/dl nmol/l μg/dl nmol/l μg/dl * Limit of Blank, or the highest value likely to be observed with a sample containing no analyte, replaces the term "analytical sensitivity." ** Proportions of false positives (α) and false negatives (β) were less than 5% and 1% respectively; based on 1578 determinations, with 214 blank and1364 low-level samples. Accuracy (Method Comparison) Accuracy was evaluated consistent with NCCLS document EP9. 23 The plots and tables show the results of a method comparison study using patient serum and urine samples from a variety of clinical categories analyzed on the VITROS ECi/ ECiQ Immunodiagnostic System using VITROS Cortisol test lot numbers below 1000 compared with those analyzed using the VITROS Cortisol test lot numbers 1000 and above. The relationship was determined by Deming regression of 16 Pub. No. GEM1401_WW_EN Version 8.0

9 INSTRUCTIONS FOR USE Performance Characteristics Cort The table also shows the results of method comparison studies 23 using patient serum, plasma and urine samples analyzed on the VITROS Immunodiagnostic System compared with those analyzed using the VITROS 3600 Immunodiagnostic System and the VITROS 5600 Integrated System. The relationship was determined by Passing and Bablok regression. 25 Serum Conventional Units Alternate Units VITROS Cortisol test (nmol/l) Lot Numbers 1000 and above VITROS Cortisol test (nmol/l) Lot numbers below 1000 VITROS Cortisol test (μg/dl) Lot Numbers 1000 and above VITROS Cortisol test (μg/dl) Lot numbers below 1000 System n Slope Lot numbers below 1000 vs. Lot numbers 1000 and above Correlation Coefficient Conventional Units (nmol/l) Range of Samples Intercept Alternate Units (μg/dl) Range of Samples Intercept vs vs Urine Conventional Units Alternate Units VITROS Cortisol test (nmol/l) Lot Numbers 1000 and above VITROS Cortisol test (nmol/l) Lot numbers below 1000 VITROS Cortisol test (μg/dl) Lot Numbers 1000 and above VITROS Cortisol test (μg/dl) Lot numbers below 1000 System n Slope Lot numbers below 1000 vs. Lot numbers 1000 and above Correlation Coefficient Conventional Units (nmol/l) Range of Samples Intercept Alternate Units (μg/dl) Range of Samples Intercept vs vs Version 8.0 Pub. No. GEM1401_WW_EN 9 of 16

10 Cort INSTRUCTIONS FOR USE Performance Characteristics Precision VITROS Immunodiagnostic System Precision was evaluated consistent with NCCLS document EP5. 26 Two replicates each of 3 freeze-dried control samples were tested on 2 separate occasions per day on at least 20 different days. An identical experiment was performed for urine after doing urine extraction pretreatment to assess precision for urine samples. The experiment was performed using 2 reagent lots on 2 different systems. The data presented are a representation of the product performance. VITROS 3600 Immunodiagnostic System and VITROS 5600 Integrated System Precision was evaluated consistent with NCCLS document EP5. 26 Two replicates each of 3 freeze-dried control samples were tested on 2 separate occasions per day on at least 20 different days. An identical experiment was performed for urine after doing urine extraction pretreatment to assess precision for urine samples. The experiment was performed using 1 reagent lot on each system. The data presented are a representation of the product performance. Serum System system 1 system System Mean Cortisol Conc. Units = nmol/l Within-run * Within-calibration ** Within-lab *** SD CV (%) SD CV (%) SD CV (%) No. Observ * Within-run (repeatability). Between Duplicate precision averaged over all runs ** Within-calibration. Total precision with weighted components of within-run, between run and between-day variation *** Within-lab. A measure of the effect of recalibration on total precision, calculated within reagent lot, using data from at least 4 calibrations system 1 system Mean Cortisol Conc. Units = μg/dl Within-run * Within-calibration ** Within-lab *** SD CV (%) SD CV (%) SD CV (%) No. Observ * Within-run (repeatability). Between Duplicate precision averaged over all runs ** Within-calibration. Total precision with weighted components of within-run, between run and between-day variation *** Within-lab. A measure of the effect of recalibration on total precision, calculated within reagent lot, using data from at least 4 calibrations No. Days No. Days 10 of 16 Pub. No. GEM1401_WW_EN Version 8.0

11 INSTRUCTIONS FOR USE Performance Characteristics Cort Extracted Urine System system 1 system System Mean Cortisol Conc. Units = nmol/l Within-run * Within-calibration ** Within-lab *** SD CV (%) SD CV (%) SD CV (%) No. Observ * Within-run (repeatability). Between Duplicate precision averaged over all runs ** Within-calibration. Total precision with weighted components of within-run, between run and between-day variation *** Within-lab. A measure of the effect of recalibration on total precision, calculated within reagent lot, using data from at least 4 calibrations system 1 system Specificity Mean Cortisol Conc. Units = μg/dl Within-run * Within-calibration ** Within-lab *** SD CV (%) SD CV (%) SD CV (%) No. Observ * Within-run (repeatability). Between Duplicate precision averaged over all runs ** Within-calibration. Total precision with weighted components of within-run, between run and between-day variation *** Within-lab. A measure of the effect of recalibration on total precision, calculated within reagent lot, using data from at least 4 calibrations Substances that do not Interfere The VITROS Cortisol test was evaluated for interference consistent with CLSI document EP7. 15 Of the compounds tested, none was found to cause a bias of >10% with the test at the concentrations indicated at cortisol concentration of nmol/l ( μg/dl). No. Days No. Days Version 8.0 Pub. No. GEM1401_WW_EN 11 of 16

12 Cort INSTRUCTIONS FOR USE Performance Characteristics Test Substance Concentration Azide 1.5 mmol/l 20 mg/dl Bilirubin 1.71 mmol/l 100 mg/dl Triolein 33.9 mmol/l 3000 mg/dl Dipyrone 3.00 mmol/l 100 mg/dl Biotin (serum) μmol/l 1 μg/dl Biotin (extracted urine) mmol/l 1000 μg/dl Intralipid * 600 mg/dl 6 g/l * Intralipid evaluated at cortisol concentrations of 20 nmol/l (0.73 μg/dl) and 675 nmol/l (24.5 μg/dl). Cross-Reactivity The cross-reactivity of the VITROS Cortisol test was evaluated by adding the following substances to a control sample containing zero analyte. Cross-reactivity was expressed as the mean result obtained for the cross reactant pool divided by the cross-reactant concentration in percentage term. Compound Concentration Tested Mean sample value of cross reactant pool µg/ml nmol/l nmol/l µg/dl Estriol 25 96, Fludrocortisone 10 26, Prednisone , Testosterone , Progesterone , α-Hydroxyprogesterone , β-cortol , β-cortolone 30 81,855 <3.00 <0.11 ND * Dexamethasone , β-Hydroxyprogesterone 10 30, α-Methylprednisolone 10 26, Deoxycorticosterone 30 86, Tetrahydrocortisol 30 81, Topiramate 100 2,946,000 <3.00 <0.11 ND * * Not Detectable. Concentration <3.00 nmol/l (<0.11 μg/dl). Mean % Crossreactivity % Cross-reactivity = Mean Result for the Cross-reactant Pool 100 Concentration of Cross-reactant Cross-Reactivity at 50% Displacement Cross-reactivity at 50% displacement was calculated from the dose-response curves obtained for the cross reactants and from the calibration curve for cortisol. The concentrations at 50% displacement of the zero calibrator for the cross-reactant and for cortisol were obtained from these curves. Compound % Cross-reactivity Prednisolone 26.8% 11-Deoxycortisol 0.95% Corticosterone 1.03% Cortisone 0.13% Concentration at 50% displacement for Cortisol % Cross-reactivity at 50% displacement = 100 Concentration at 50% displacement for Cross-reactant 12 of 16 Pub. No. GEM1401_WW_EN Version 8.0

13 INSTRUCTIONS FOR USE References Cort References 1. Borth R. Problems related to the chemical assay of corticosteroids in human body fluids. Acta Endocrinologica 22: ; Harris GW, Donovan BT, Best OH, Taylor NB (eds). The adrenal gland. The Physiological Basis of Medical Practice. ed ; James VHT, Landon J (eds). Control of corticosteroid secretion current views and methods of assessment. Recent Advances in Endocrinology. ed. 8: 50 94; Wagner RK. Extracellular and intracellular binding proteins. Properties, discrimination, assay and clinical application. Acta Endocrinologica. Supplement 218, vol.88: 3 73; Hellman L et al. Cortisol is secreted episodically by normal man. Journal of Clinical Endocrinology and Metabolism. 30: ; Sherman B, Wysham C, Pfohl B. Age-related changes in the circadian rhythm of plasma cortisol in man. Journal of Clinical Endocrinology and Metabolism. 61: ; Summers M et al. Luminogenic Reagent Using 3-Chloro 4-Hydroxy Acetanilide to Enhance Peroxidase/Luminol Chemiluminescence. Clin Chem. 41:S73; CLSI. Protection of Laboratory Workers from Occupationally Acquired Infections; Approved Guideline - Fourth Edition. CLSI document M29-A4. Wayne, PA: Clinical and Laboratory Standards Institute; Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/ Calam RR. Specimen Processing Separator Gels: An Update. J Clin Immunoassay. 11:86 90; CLSI. Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; Approved Standard Sixth Edition. CLSI document H3-A6 (ISBN ). CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania , USA NCCLS. Procedures and Devices for the Collection of Diagnostic Capillary Blood Specimens; Approved Standard Fifth Edition. NCCLS document H4-A5 (ISBN ). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania , USA NCCLS. Urinalysis and Collection, Transportation, and Preservation of Urine Specimens; Approved Guideline Second Edition. NCCLS document GP16-A2 (ISBN ). NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania , USA CLSI. Statistical Quality Control for Quantitative Measurements: Principles and Definitions; Approved Guideline - Third Edition. CLSI document C24-A3 [ISBN ]. CLSI, 940 West Valley Road, Suite 1400, Wayne, PA USA, NCCLS. Interference Testing in Clinical Chemistry; Approved Guideline. NCCLS document EP7-A (ISBN ). CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania , USA Scientific Committee on Food. Opinion of the Scientific Committee on Food on the Tolerable Upper Intake Level of Biotin. European Commission, SCF/CS/NUT/UPPLEV/55 Final, Brussels, Levinson SS. The Nature of Heterophilic Antibodies and Their Role in Immunoassay Interference. J Clin Immunoassay. 15: ; Kricka, LJ. Human Anti-Animal Antibody Interferences in Immunological Assays. Clin Chem 45: ; Young DS. Effects of Drugs on Clinical Laboratory Tests. ed. 4. Washington, D.C.: AACC Press; Friedman RB, Young DS. Effects of Disease on Clinical Laboratory Tests. ed. 3. Washington, D.C.: AACC Press; Tryding N, Tufvesson C, Sonntag O (eds). Drug Effects in Clinical Chemistry. ed. 7. Stockholm: The National Corporation of Swedish Pharmacies, Pharmasoft AB, Swedish Society for Clinical Chemistry; NCCLS. Protocols for Determination of Limits of Detection and Limits of Quantitation; Approved Guideline. NCCLS document EP 17-A (IBSN ). CLSI, 940 West Valley Road, Suite 1400, Wayne Pennsylvania USA, NCCLS. Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline-Second Edition. NCCLS document EP9-A2(ISBN ). CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania , USA Deming WE, Statistical Adjustment of Data. New York, NY: John Wiley and Sons; NCCLS. Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline-Second Edition. 25. Passing H, Bablok W. A New Biometrical Procedure of testing the Equality of Measurements from Two Different Analytical Methods. J. Clin Chem Biochem. 21: , NCCLS. Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline Second Edition. NCCLS document EP5-A 2(ISBN ). CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania USA, Version 8.0 Pub. No. GEM1401_WW_EN 13 of 16

14 Cort INSTRUCTIONS FOR USE Glossary of Symbols Glossary of Symbols Revision History Date of Revision Version Description of Technical Changes* Reagent Pack Storage and Preparation: 3600/ Lot 1738 and above: changed open stability to 7 weeks : removed lot number Warnings and Precautions: changed Kathon to Kathon or ProClin 200 Limit of Detection: changed 1% to 5% Updated legal manufacturer address 14 of 16 Pub. No. GEM1401_WW_EN Version 8.0

15 INSTRUCTIONS FOR USE Revision History Cort Date of Revision Version Description of Technical Changes* Prescription Use Statement added Warnings and Precautions: added reference updated Hazard and Precaution Statements to align with the new Safety Data Sheets added Globally Harmonized Symbol to comply with the Classification, Labelling and Packaging (CLP) Regulations Measuring (Reportable) Range: removed lot numbers References: updated M29 added reference Glossary of Symbols: added Globally Harmonized Symbols to comply with the Classification, Labelling and Packaging (CLP) Regulations Calibrator Storage and Preparation: clarification of the frozen storage temperature Glossary of Symbols: added Date of Manufacture Glossary of Symbols: updated wording Brazilian address: updated Reagent Pack Storage and Preparation - (Lot 1250 and above): changed open stability to 8 weeks New format that combines the following into one document: Cortisol Reagent Pack (GEM1401_EN_XUS), version 1.0 Cortisol Reagent Pack (GEM1401_EN_US), version 1.0 Cortisol Calibrators (GEMC401_EN_WW), version 1.0 Added information for the VITROS 3600 Immunodiagnostic System and the VITROS 5600 Integrated System Updated risk and safety statements Reagent Pack Storage and Preparation (Lot 1250 and above): changed open stability to 6 weeks Measuring (Reportable) Range: updated lower limit based on limit of quantitation for the VITROS Immunodiagnostic and VITROS Integrated Systems Substances that do not Interfere: added Intralipid Analytical Sensitivity: removed section References: updated Glossary of Symbols: updated * The change bars indicate the position of a technical amendment to the text with respect to the previous version of the document. When this Instructions For Use is replaced, sign and date below and retain as specified by local regulations or laboratory policies, as appropriate. Signature Obsolete Date Version 8.0 Pub. No. GEM1401_WW_EN 15 of 16

16 Cort INSTRUCTIONS FOR USE Revision History Conditions of supply: all supplies are made subject to the standard terms and conditions of Ortho-Clinical Diagnostics or its distributors. Copies of these are available on request. Distributed in the US by: Ortho-Clinical Diagnostics, Inc. 100 Indigo Creek Drive Rochester, NY Ortho-Clinical Diagnostics Felindre Meadows Pencoed Bridgend CF35 5PZ United Kingdom VITROS is a trademark of Ortho-Clinical Diagnostics, Inc. Ortho-Clinical Diagnostics, Inc., of 16 Pub. No. GEM1401_WW_EN Version 8.0