Cortisol Serum, Plasma and Urine Protocol

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is controlled by hypothalamic corticotropin-releasing hormone (CRH). CRH, ACTH ... Serum Samples (n=74) were run on the IDS-iSYS at IDS PLC. and on the.

P023 A fast and fully automated chemiluminescence immunoassay for the determination of cortisol in serum, plasma, urine and saliva Grimminger P1, Osswald A1, Reincke M1, Roffe CM2, Bidlingmaier M1 1

Endocrine Research Unit, Medizinische Klinik und Poliklinik IV, Ludwig Maximilians University, Munich, Germany; 2 Immunodiagnostic Systems (IDS), Boldon, UK

Purpose and Methods

Introduction

Current guidelines for the diagnosis of hypercortisolism recommend the measurement of cortisol in blood following stimulation or suppression stress tests, but also the measurement of integrated 24 hours cortisol secretion in urine or late night salivary cortisol. More recently, measurement of cortisol has also been proposed as a marker of selectivity of canulation of the adrenal veins during adrenal vein sampling procedures. The latter, however, requires the availability of a very rapid cortisol measurement procedure. Our aim was to develop an automated cortisol assay with high specificity based on a high affinity monoclonal antibody allowing very accurate and rapid measurement of cortisol concentrations in different specimens – namely serum, plasma urine and saliva on one platform. We present initial results from the development study and the initial validation of the new cortisol assay. We also compare the results to those of established and widely used immunoassays in patients samples and in samples from external quality assessment schemes. Finally, we used our routine method and the new assay to asses cortisol concentrations in samples from adrenal vein samples where high levels of potentially cross reacting steroids and extremely high concentrations of cortisol can be found.

Results Protocol In the assay, cortisol in the sample competes with the biotinylated cortisol for the binding to a monoclonal anti-cortisol-antibody/acridinium conjugate in the first incubation. Streptavidin coupled magnetic particles are then added. After a further incubation and a wash step, the bound anti-cortisolacridinium is measured whereby the chemiluminescence generated is inversely proportional to the cortisol concentration in the sample. The time to first result is less than 8 minutes.

Calibration and Assay Range Calibration was performed by using a 7 point calibrator set prepared with a buffer-based matrix. The IDS-iSYS Cortisol assay had an assay range up to 15.9µg/dL for Saliva samples and 79.5µg/dL for Serum, Plasma and Urine samples. LOQ for Saliva was measured at 0.08µg/dL and 0.8µg/dL for serum, plasma and urine. ED50 is 0.35µg/dL. Fig 2. Mean Calibration Curve of IDS-iSYS Cortisol Assay.

Fig 1. Schematic diagram of the IDS-iSYS Cortisol assay

IDS iSYS Mean Cortisol Standard Curve.

100%

2 min

+

Cortisol in the sample and calibrator

Biotinylated Cortisol

Rapid cortisol assessment during adrenal vein sampling Adrenal vein sampling (AVS) was conducted to identify the source of excess aldosterone production in patients with primary aldosteronism (Conn’s Syndrome). To this purpose, EDTA Plasma samples were obtained in the department of radiology at the locations indicated in figure 7. Immediately after collection, plasma samples were centrifuged and analyzed for cortisol concentrations by our routine method (Liaison, Diasorin) cortisol assay. On average, information on the selectivity index could be transmitted to the radiologist after 26 minutes. The same samples were re-analysed using the newly developed IDS iSYS cortisol assay, where results were available after 10 minutes (mean). Fig 7. Adrenal vein sampling

%B/Bo

+

75%

Acridinium conjugated anti-Cortisol MAb

50%

Rapid cortisol assessment during AVS is performed to asses the positioning of the catheter. Cortisol levels must be at least 2x higher in the adrenal veins than in the peripheral vessels to assume “selectivity”. Especially on the right side (adrenal vein leads into v. cava) success rates are poor. Rapid feedback from cortisol assessments during the procedure has been shown to increase success rates dramatically.

Cortisol and Biotinylated Cortisol attached to Acridinium conjugated anti-Cortisol Mab 25%

2 min

Wash & Read

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0% 0,01

0,1

1

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Cortisol (ug/dL)

Streptavidin coated magnetic particles

Immunocomplex attached to Streptavidin coated magnetic particles

Cortisol Serum, Plasma and Urine Protocol On-board pre-dilution. 50µL Sample + 200µL Assay buffer 25µL Cortisol-Biotin 100µL Anti-cortisol-acridinium 100µL Pre-diluted sample Incubate 2 minutes at 37°C 20µL Streptavidin coated Magnetic Particles

Cortisol Saliva protocol 25µL Cortisol-Biotin 100µL Anti-cortisol-acridinium 100µL Sample Incubate 2 minutes at 37°C 20µL Streptavidin coated Magnetic Particles Incubate 2 minutes at 37°C

Correlation Serum Samples (n=74) were run on the IDS-iSYS at IDS PLC. and on the Roche Cobas at Semmelweis University Hospital, Budapest, Hungary. The Urine samples (n=25) were run on the IDS-iSYS and the Siemens RIA CoatA-Count (Ref: PITKCO) at IDS PLC. Saliva samples (n=24) were run on the IDS-iSYS and IBL EIA (Ref: RE52611) at IDS PLC. Serum and Plasma samples from two external quality assessment UK NEQAS (n=15) and the German DGKL (n=15) were run on the IDS-iSYS and correlated against the Roche Cobas. Fig 3. Serum Cortisol: IDS-iSYS vs. Roche Cobas.

Fig 4. Urinary Cortisol IDS-iSYS vs. Siemens CAC

Wash and Read

45

70

y = 1,03x + 0,35 R² = 0,96

Incubate 2 minutes at 37°C 60

Wash and Read

y = 0,83x + 1,08 R² = 0,97

40

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50

Fig 8. Plasma Cortisol IDS-iSYS Vs Diasorin Liaison

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IDS iSYS (µg/dL)

IDS iSYS (µg/dL)

Specificity A set of serial dilution was performed on the compounds listed below and assayed along side the calibration curve. The cross reactivity was calculated using the concentration obtained from the ED50.

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IDS iSYS vs. Diasorin Liaison (n=127 AVS)

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Roche (µg/dL)

Compound

% Cross reactivity

Corticosterone

17.5%

Cortisone

14.0%

21-Deoxycortisol

19.4%

Dexamethasone

0.44%

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35

40

45

Fig 6. Cortisol IDS-iSYS DGKL and NEQAS Samples 50

y = 0,79x - 0,01 R² = 0,99

10

25

Siemens Coat-A-Count (µg/dL)

Fig 5. Saliva Cortisol IDS-iSYS vs. IBL EIA. 12

20

y = 0,98x + 0,06 R² = 0,99 40

0.12%

6ß-Hydroxycortisol

0.04%

17α-Hydroxyprogesterone 6α-Methylprednisolone Progesterone Prednisone

1.40% 0.09% 0.23% 18.4%

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6

4

14.0%

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2000

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20

including samples with extremely high cortisol concentrations (n=174)

2500

10

y = 0,701x + 2,5724 R² = 0,9183

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0 0

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Liaison Cortisol in plasma [µg/dL]

German DGKL 10

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UK NEQAS

0

Linear (German DGKL)

0

Linear (UK NEQAS) 0 0

Prednisone

y = 0,90x + 1,86 R² = 0,96

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iSYS (µg/dL)

6α-Hydroxycortisol

50

iSYS Cortisol in plasma [µg/dL]

0

iSYS Cortisol in plasma [µg/dL]

Table 1. IDS-iSYS Cortisol Analytical specificity

y = 0,9646x + 0,7015 R² = 0,894

60

0

IDS iiSYS (µg/dL)

10. Jahrestagung der DGKL, 23. - 26. Oktober 2013, Dresden

Cortisol is a steroid-hormone synthesized in multiple steps from cholesterol in the zona fasciculate of the adrenal gland. Its secretion is controlled by the pituitary adrenocorticotropic hormone (ACTH), which in turn is controlled by hypothalamic corticotropin-releasing hormone (CRH). CRH, ACTH and Cortisol are regulated by a negative feedback-loop. The secretion of ACTH and Cortisol is subject to a strong circadian rhythm showing the highest levels in the morning and a nadir in the late evening or night. Cortisol is also an important “stress-hormone”, and released in response to physical and psychological stress. The primary function of cortisol is to increase the level of blood sugar through gluconeogenesis, suppressing the immune system and aiding fat, sugar and carbohydrate metabolism. Another long-term effect is also the decrease of bone formation. Chronic excess of cortisol (Cushing’s Syndrome) is associated with truncal obesity, facial fullness, hypertension, diabetes or glucose intolerance, gonadal dysfunction, mood disorders and osteoporosis, while cortisol deficiency (adrenal insufficiency, Addisons disease) is a life threatening condition associated with tiredness, weakness, hypotension, mental depression, anorexia and weight loss. Clinical signs are not specific so measuring cortisol levels is mandatory for the diagnosis of these conditions.

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6 IBL EIA (µg/dL)

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Liaison Cortisol in plasma [µg/dL]

Roche (µg/dL)

Results and Discussion Results: The assay range covering all matrices is 0.05-75µg/dL with an analytical sensitivity of 0.02µg/dL. The results for serum and plasma (test samples of UK NEQAS, DGKL and patient samples) were correlated against commercially available assays for serum and plasma (Diasorin Liaison, Roche Cobas). They showed also excellent correlation to mass spectrometry values of the DGKL samples. For urine and saliva a good correlation to the IBL assays was observed. Conclusion: The fully automated IDS-iSYS cortisol assay potentially presents a new, accurate and reliable immunoassay for all commonly used sample types. The extremely short time to first result makes the assay suitable also for use during diagnostic procedures like adrenal vein sampling.

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