Measurement of salivary cortisol levels by an automated electrochemiluminescence immunoassay method (ECLIA): Evaluation of analytical performanceC. Acevedo, M. Morini, M.Gomez, M.E.Almagro, M.G.RopelatoLaboratorio de Medicina S.A. Olaya 1644 CABA e-mail: endocrinologia@labmedicina.com

BACKGROUNDThe diagnosis and treatment of Cushing’s Syndrome represents a challenge in clinical endocrinology as the features of endogenous hypercortisolism are common to other clinical conditions. The lack of circadian rhythm in adrenal cortical secretion is one of the hallmarks of this disease. Salivary cortisol reflects the unbound, biologically active form of serum cortisol. Consequently, the measurement of late evening salivary cortisol is especially useful for evaluating ambulatory patients and for the pediatric population, given it is a noninvasive method

OBJECTIVETo evaluate the analytical performance of the ECLIA method for the measurement of salivary cortisol

MATERIALS Y METHODS

An automated immunoassay with electrochemiluminescent signal (Modular E170 Roche) was used

Allowable total error (TEa) was established as 3CVi = 20% Samples of saliva were obtained from healthy volunteers (13 women and 7 men) between 26 and 45 years of age. BMI<25 kg/m² .These were collected at 8:00, 15:00 and 23:00 hours. Precision: repeatability (CVr) and intermediate precision (CVi) of two levels of concentration were evaluated with pools of samples of saliva (P1 and P2) according to EP15-A2 CLSI protocol

P1: 8.3 nmol/l P2: 16.6 nmol/l

Data were compared with manufacturer´s claims The limit of quantification ( LoQ), defined as the lowest concentration that can be quantified with an intermediate precision, CVi <20%, was determined based on Spenser et al. (Demmers LM and Spencer LA (eds.) National Academy of Clinical Biochemistry Support for the diagram and monitoring of thyroid disease. 2003,

13:4-126) Five concentrations of saliva near the LoQ claimed were run for 10 days The reference interval (RI) of 8:00 am and 15:00 pm proposed by the manufacturer was ascertained following C28 A3 CLSI protocol. The 95 percentile was estimated for the 23:00 hour samples as this RI was not reported as product information Linearity was verified across 0.74 – 17.00 nmol/L, an interval representing the lower zone of the measuring range ( 0.50 – 1750 nmol/l)

Reference Interval Histogram

15:00 hours8:00 hours

RESULTS

CONCLUSIONS The assessment of the ECLIA analytical performance enabled us to verify the precision and reference interval specifications for salivary cortisol claimed by the manufacturer .

The LoQ obtained allowed us to ensure an adequate performance in the clinical decision levels.

LINEARITY

FUTURE OBJECTIVES Confirming our preliminary results of the reference interval in the 23:00 hour samples will be of great value in the evaluation of the hypercortisolism disorders.

Our mayor goal is to establish the laboratory´s pediatric RI for salivary cortisol.

CSOL 8 hs Csol 15 hs Csol 23 hs0,00

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Cortisol circadian rhythm

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  ROCHE Laboratorio de Medicina

Repeatability

P1 CVr 4.7% 4.0%

P2 CVr 6.4% 4.4%

Intermediate Precision

P1 CVi 10.0% 6.7%

P2 CVi 9.5% 5.6%

LoQ < 2.0 nmol/l < 2.2 nmol/l

R.I 8:00 hs. 1.9 – 19.1 nmol/l Verified

R.I 15:00 hs. 1.9 – 11.9 nmol/l Verified

P 95% 23:00 hs. Not Reported < 5.0 nmol/l (1)

Clinical Linearity - 0.74 - 17.00 nmol/l

(1) Preliminary Data

Range Tested: 0.74 to 17.0 nmol/L

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Cort 8hs Cort 15 hs Cort 23hs

Cort 8hs Cort 15 hs Cort 23hs