LIAISON ACTH (REF )

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1 For Professional Use Only! DiaSorin S.p.A. Strada per Crescentino Saluggia (Vercelli)-Italy Tel Fax LIAISON ACTH (REF ) 1. INTENDED USE The LIAISON ACTH assay uses chemiluminescent immunoassay (CLIA) technology for the in vitro quantitative determination of Adrenocorticotropic hormone in human EDTA-plasma specimens (stored frozen). The determination of ACTH in conjunction with other laboratory tests and clinical findings can help in investigating adrenal dysfunction in humans. The test has to be performed on the LIAISON Analyzer. 2. SUMMARY AND EXPLANATION OF THE TEST Adrenocorticotropic hormone or corticotrophin (ACTH) is a 39 amino acid polypeptide secreted by the anterior pituitary gland 1, 2. It stimulates the adrenal cortex to secrete glucocorticoids like Cortisol and exerts some control over secretion of Aldosterone, the other major steroid hormone secreted by the adrenal cortex. The hypothalamus controls pituitary ACTH secretion by means of corticotrophin releasing hormone (CRH), a 41 amino acid peptide released in response to pain, anxiety, and stress via neurotransmitters. CRH itself is inhibited by glucocorticoids, making it a part of classical negative feedback loop 2. Cortisol also exerts negative feedback control on the secretion of ACTH at the pituitary gland and hypothalamic levels. Corticotrophin secretion is also under circadian control by a number of factors including light. Conditions that have been demonstrated to alter the normal diurnal rhythm include Cushing s syndrome and ectopic ACTH secretion as well as physiologic stress or surgery 3, 4. Diurnal variations of secretion are well-known: the ACTH secretory bursts increase in frequency after three to five hours of sleep and are maximal prior to awakening and the hour after. Because of this diurnal variation it is used to draw plasma ACTH samples between a.m. Due to the lack of diurnal rhythm in Cushing s disease, the discrimination of patients from normal individuals may be best made on samples obtained in the evening (10.00 p.m. to midnight). Within the pituitary gland, ACTH is produced in a process that also generates several other hormones. The precursor of ACTH is a large protein called propiomelanocortin (POMC) synthesized and proteolytically cleaved into fragments that include also lipotropin (precursor of beta-endorphin and met-enkephalin) and melanocyte-stimulating hormone (MSH). Plasma ACTH determination is useful in diagnosing disorders of the hypothalamic-pituitary-adrenal system. Increased ACTH levels are found in Pituitary Cushing`s disease (secondary hypercortisolism) Addison s disease (hypocortisolism) Autonomous ACTH producing tumours (ectopic ACTH syndrome) Lowered ACTH levels are found in Adrenal adenoma (primary Cushing s syndrome) Secondary adrenal insufficiency The prevalent disorder involving glucocorticoids is secondary Cushing s syndrome or hyperadrenocorticism, in which ACTH levels are increased. The most common causes of Cushing s syndrome, when administration of glucocorticoids for therapeutic purposes is excluded, are: bilateral adrenal hyperplasia (due to pituitary ACTH hypersecretion, called Cushing s disease), pituitary adenoma or corticotrophic hyperplasia. Laboratory diagnosis of Cushing s disease is supported by stimulation or suppression tests like: high-dose dexamethasone administration (suppression of ACTH and Cortisol secretion), low-dose dexamethasone, increased Cortisol response to metyrapone and normal or elevated ACTH levels. Another condition in which ACTH levels are increased is the Nelson s syndrome; this is an almost rare syndrome occurring in 15-25% of people who undergo removal of the adrenal glands for Cushing s disease. It is characterized by abnormal hormone secretion, enlargement of the pituitary gland and development of large and invasive adenomas. In addition to adrenocortical hyperfunction, ACTH levels can be affected by adrenal insufficiency (Hypocortisolism). In these cases Cortisol determination by itself is not sufficient in differentiating primary causes of adrenal insufficiency (due to Addison s disease) from secondary causes. ACTH determination is useful in these cases to better understand the real type of the disease. Disease Cortisol level ACTH level Cushing's disease (pituitary tumour making ACTH) high high "Ectopic" ACTH (ACTH secreted by a tumour outside the pituitary) high high Adrenal tumour high low Addison s disease (adrenal damage) low high Hypopituitarism low low 3. PRINCIPLE OF THE PROCEDURE The method for the quantitative determination of ACTH (1-39) is a sandwich chemiluminescence immunoassay. A specific mouse monoclonal antibody (N-terminal) is coated on the magnetic particles (solid phase); another monoclonal antibody (directed toward the C-terminal fragment of ACTH) is linked to an isoluminol derivative (isoluminol-antibody conjugate). During the incubation, ACTH (1-39) present in calibrators, samples or controls binds to the solid phase monoclonal antibody, and subsequently the antibody conjugate reacts with ACTH (1-39) already bound to the solid phase. After incubation, the unbound material is removed with a wash cycle. Subsequently, the starter reagents are added and a flash chemiluminescence reaction is thus induced. The light signal, and hence the amount

2 of isoluminol-antibody conjugate, is measured by a photomultiplier as relative light units (RLU) and is indicative of ACTH concentration present in calibrators, samples or controls. 4. MATERIALS PROVIDED The order of reagents reflects the layout of containers in the reagent integral. Reagent Integral for ml Magnetic particles suspension: magnetic particles, coated with anti-acth, monoclonal (mouse). 23 ml Tracer conjugate : anti-acth, labelled with isoluminol derivative, monoclonal (mouse). Included with Integral 3 x 1.5 ml Calibrator low, lyophilized, human ACTH in human serum albumin solution. 3 x 1.5 ml Calibrator high, lyophilized, human ACTH in human serum albumin solution. Conjugate and magnetic particles are provided ready-to-use. Calibrators are provided lyophilized. Materials required but not provided Additionally required materials LIAISON Module (REF ). LIAISON ACTH controls, low and high (REF ). LIAISON Starter Kit (REF ). LIAISON Cleaning Kit (REF ). LIAISON Light Check (REF ). LIAISON ACTH/Cortisol diluent (REF ). LIAISON Wash/System Liquid (REF ). LIAISON Waste Bags (REF ). 5. WARNINGS AND PRECAUTIONS For in vitro diagnostic use. All materials used to produce the components provided in this kit have been tested for the presence of HBsAg, anti-hcv, anti-hiv-1, anti-hiv- 2 and found to be non-reactive. As, however, no test method can offer absolute assurance that pathogens are absent, all specimens of human origin should be considered potentially infectious and handled with care. 6. SAFETY PRECAUTIONS Do not eat, drink, smoke or apply cosmetics in the assay laboratory. Do not pipette solutions by mouth. Avoid direct contact with all potentially infectious materials by using protective clothing such as lab coats, protective glasses and disposable gloves. Wash hands thoroughly at the end of each assay. Avoid splashing or forming an aerosol. Any reagent spills should be washed with a 5% sodium hypochlorite solution and disposed of as though potentially infectious. All samples, biological reagents and materials used in the assay must be considered potentially able to transmit infectious agents. They should therefore be disposed of in accordance with the prevailing regulations and guidelines of the agencies holding jurisdiction over the laboratory, and the regulations of each Country. 7. REAGENT PREPARATION REAGENT INTEGRAL Before removing the seals from the containers, gently and carefully shake the reagent integral horizontally. Avoid formation of foam. Remove the seal completely from each container and turn the thumb wheel at the bottom of the magnetic particle container to and fro until the suspension turns brown. This procedure initiates resuspension of magnetic particles. Carefully wipe the surface of each septum to remove residual liquid. Then, place the integral into the reagent area of the Analyzer with the barcode label facing left and let it stand for 30 minutes before using. The Analyzer automatically stirs and completely resuspends the magnetic particles. Follow the Analyzer Operator s Manual to load the specimens and start the run. CALIBRATORS LIAISON ACTH calibrators are supplied lyophilized. - Reconstitute with 1.5 ml deionized or distilled water. - Allow the vials to stand for 10 minutes at approximately C. - Mix thoroughly by gentle inversion, avoid foaming. Once reconstituted refer to paragraph 8 to store the calibrators. For details on the use of the calibrators, refer to the LIAISON Operator s Manual. Concentrations of ACTH in the calibrators are printed on the vial labels and coded in the barcode of the vials. CONTROLS Refer to the LIAISON ACTH Control Set instructions for use section for proper preparation and handling instructions. 8. REAGENT STORAGE AND STABILITY REAGENT INTEGRAL - Sealed: Stable at 2-8 C until the expiry date. - Opened on board or at 2-8 C: Minimum stability two weeks. After this period, it is still possible to keep on using the reagent integral provided that the controls are found within the expected ranges. - Use always the same LIAISON Analyzer for a reagent integral already opened. - Do not freeze. - Keep upright for storage to facilitate later proper resuspension of magnetic particles. - Use storage rack provided with the LIAISON Analyzer for upright storage of reagent integral. - Keep away from direct light. CALIBRATORS - Lyophilized: Stable at 2-8 C until the expiry date.

3 - Reconstituted: Stable for three days when properly stored at 2-8 C. - Frozen: Aliquots can be stored at -20 C for up to one month. The ACTH molecule has limited stability against heat stress in liquid solutions: do not leave the reconstituted calibrators at room temperature longer than the time required to process them on the LIAISON. Immediately after complete reconstitution it is possible to aliquot the calibrators in plastic or siliconized tubes and deep-freeze them. After thawing the calibrators must be used up in the same day. The minimum volume of the aliquot is 600 µl (450 µl calibrator µl dead volume). Only one freeze and thaw cycle is allowed for each aliquot. During handling, use appropriate precautions to avoid bacterial contamination of calibrators. 9. SPECIMEN COLLECTION AND PREPARATION The ACTH molecule degrades rapidly in the collected specimen; the following recommendations for handling the samples must be followed very carefully. The only sample material validated is human EDTA-plasma stored frozen. Collect blood by venipuncture, in siliconized glass tubes, vacutainers (violet cap) or equivalent, containing EDTA as anticoagulant. Avoid haemolysis. Put tubes immediately in an ice bath, keep cool. Centrifuge not later than 120 minutes after drawing, using a refrigerated centrifuge, and immediately aliquot and deep-freeze (-20 C or below) in plastic or siliconized glass tubes. The minimum volume of the aliquot is 300 µl. Carefully thaw before testing, mix the thawed samples and check for and remove air bubbles before assaying. Thawed samples kept at 2-8 C must be used within six hours. Grossly haemolyzed or lipaemic samples as well as samples containing particulate matter or exhibiting obvious microbial contamination should not be tested. Do not use clotted samples. Avoid repeated freeze and thaw cycles. Dispose of any left aliquot volume. The minimum volume required for a single determination is 300 µl specimen (150 µl specimen µl dead volume). 10. CALIBRATION Assay of calibrators contained in the reagent integral allows the Analyzer to recalibrate the stored master curve, as indicated via the barcodes on the reagent integral label. The Analyzer should be calibrated in triplicate whenever one of the following conditions occurs: - A new lot of Starter Kit is used. - After 1 week and/or each time a new Integral is used (recommendation). - The Analyzer has been serviced. - Control values lie outside the expected ranges. 11. ASSAY PROCEDURE Strict adherence to the Analyzer Operator s Manual ensures proper assay performance. Each test parameter is identified via the barcodes on the reagent integral label. In case of malfunction of the barcode reader, the data can be entered manually. For details, refer to the Analyzer Operator s Manual. The Analyzer operations are as follows: 150 µl Calibrators, controls or specimens µl Coated magnetic particles µl Tracer conjugate. 20 min Incubation followed by a wash cycle. 3 s Measurement. 12. QUALITY CONTROL Quality control must be performed (a) at least once per day of use, (b) whenever a new reagent integral is used, (c) whenever the kit is calibrated, or in agreement with guidelines or requirements of local regulations or accredited organizations, (d) whenever a new lot of Starter Reagent is used, (e) to assess adequacy of performance of the open integral beyond two weeks. Warning: LIAISON controls should be run in singlicate to monitor the assay performance. Control values must lie within the expected ranges: whenever controls lie outside the expected ranges, calibration should be repeated and controls retested. If control values obtained after successful calibration lie repeatedly outside the predefined ranges, the test should be repeated using a freshly reconstituted control vial. If control values lie outside the expected ranges, patient results must not be reported. The performance of other controls should be evaluated for compatibility with this assay before they are used. Appropriate value ranges should then be established for quality control materials used. 13. INTERPRETATION OF RESULTS The Analyzer automatically calculates ACTH concentrations for the unknown samples in. For details, refer to the Analyzer Operator s Manual. Measuring range: The Analyzer directly calculates ACTH concentration up to corresponds to pmol/l. Basal reference range: To assess ACTH reference range a clinical study was performed in 589 prospectively collected samples from European subjects sent to the laboratory for ACTH testing. The subjects (males = 270, females = 313, unknown = 6) were classified as normal when tested by a reference method. The study yielded a median value of 16.3 and basal ACTH values ranging between 4.7 and 48.8 (5th and 95th percentile respectively). Each laboratory should establish its own basal reference range.

4 Clinical samples: A clinical study was also performed in 51 pathological samples; statistical analysis of the normal and pathological samples is shown in the following table. Correlation of the clinical levels obtained in a total of 640 samples collected from normal subjects and patients with pituitary-adrenal diseases is illustrated in the following graph. Clinical condition N. Dexamethasone suppression test (ST) Mean, S.D., 2.5th percentile, Median, (50th percentile) 97.5th percentile, Min. value, Max. value, Addison s disease (AD) Ectopic ACTH syndrome (ES) Cushing s syndrome (CS) Nelson s syndrome (NS) Sinus petrosus catheterization (SPC) Normal subjects (N) LIMITATIONS OF THE PROCEDURE - The reagents should be used only in the LIAISON System. - Single components of the reagent integral should not be removed from the integral. - This kit must not be used after the expiry date printed on the package label. - A skillful technique and strict adherence to the instructions are necessary to obtain reliable results. - Bacterial contamination or heat inactivation of the specimens may affect the test results. - A non-pathological result does not always rule out the presence of adrenal dysfunction and should be interpreted together with other diagnostic procedures. - Test results are reported quantitatively. However, diagnosis of an adrenal disease should not be based on the result of a single test, but should be determined in conjunction with clinical findings in association with medical judgement. Any therapeutical decision must also be taken on a case-by-case basis. - The LIAISON ACTH assay has been developed for the determination of the analyte in its intact and unaltered state. Degradation of the molecule into fragments may alter antibody binding characteristics and affect final results. Such samples may exhibit discordant results between different assays, as the effect of such altered states are particular to each defined antibody reagent used. - Although HAMA-neutralizing agents are added, extremely high HAMA (human anti-mouse antibodies) concentrations may occasionally influence results. - Samples containing ACTH levels above the measuring range may be prediluted with the LIAISON ACTH/Cortisol diluent (REF ). Warning: The assay has been developed to test frozen EDTA-samples and the basal reference range, and all performance data provided have been established on frozen EDTA-plasma samples. In case fresh samples will be used, each laboratory should establish its own range of expected values. As an example, we report the correlation found between fresh and frozen samples run with the LIAISON ACTH method in the study where the Basal Reference Range has been established (see Interpretation of Results).

5 y = x R = N = 72 Fresh samples, Blood samples kept cooled up to +15 minutes. Clinical samples with very high ACTH levels, above 20,000 were not tested. Literature reports 1 cases of Nelson s syndrome with extremely high concentration of ACTH, in excess of 20,000. If such pathology is suspected, values found within the assay measuring range should be carefully evaluated to exclude HDH effect (see paragraph 15.4) and re-testing after sample dilution should be performed. 15. SPECIFIC PERFORMANCE CHARACTERISTICS Analytical specificity Analytical specificity may be defined as the ability of the assay to accurately detect specific analyte in the presence of potentially interfering factors in the sample matrix (e.g., haemolysis, lipaemia, bilirubinaemia). Interference. Controlled studies of potentially interfering substances or conditions showed that the assay performance was not affected by concentrations of bilirubin < mg/ml, haemoglobin < 500 mg/dl or triglycerides < 12.5 mg/ml. Cross-reactions. The presence of the following potentially cross-reactive molecules did not show any interference in the assay. Compound Spiked amount, % Cross-reactivity ACTH (1-10) undetectable ACTH (1-24) ACTH (18-39) ACTH (11-24) Alpha-MSH Beta-MSH Beta-Endorphin Somatostatin Neurotensin 9800 < 0.01 Enkephalin 9800 < Precision Different samples, containing different concentrations of ACTH, were assayed to estimate repeatability and reproducibility of the assay (i.e., within- and between-assay variability). The tests were performed in two sites, in-house and in an independent laboratory. In-house repeatability. Forty replicates were performed in the same run to evaluate repeatability. In-house reproducibility. Twenty replicates were performed in different days with two different lots of integral to evaluate reproducibility. The precision at the external site was determined according to NCCLS (National Committee for Clinical Laboratory Standards) guidelines. In-house External site Intra-assay variation Inter-assay variation Mean value () CV (%) Mean value () CV (%) Intra-assay variation Inter-assay variation Mean value () CV (%) Mean value () CV (%) (5) (5) (5) The results refer to the groups of samples investigated and are not guaranteed specifications, as differences may exist between laboratories and locations.

6 15.3. Trueness The assay trueness has been checked by the dilution and recovery tests. Dilution test. Plasma samples containing high ACTH concentrations were tested as such and after serially diluting with the specimen diluent. ACTH concentrations measured versus expected were analyzed by linear regression. The correlation coefficients (r) ranged from to Dilution factor Measured value () Expected value () Recovery (%) 1: : : : Recovery test. Plasma samples spiked with ACTH (1-39) were tested to evaluate the recovery of the LIAISON ACTH assay. Original concentration of sample 1: 6.76 and of sample 2: Measured value Expected value Measured value Expected value () () Recovery (%) () () Recovery (%) Sample 1 Sample High-dose hook effect The high-dose hook (HDH) was determined by addition of ACTH (1-39) to human plasma pools to a maximum of 148,000. Whenever samples containing extremely high analyte concentrations are tested, the HDH can mimic concentrations lower than real. Analysis of HDH was evaluated by testing five high-concentration ACTH-spiked samples. All samples resulted in calculated concentration values above the measuring range, indicating no sample misclassification Analytical and functional sensitivity Analytical sensitivity is defined as the minimum detectable dose distinguishable from zero by two standard deviations. LIAISON ACTH analytical sensitivity is < 1.6. Functional sensitivity is defined as the concentration at which the inter-assay coefficient of variation (CV) exceeds 20%. LIAISON ACTH functional sensitivity is 3.8 (determined in-house) and 7.0 (determined in the external site). Accuracy of the LIAISON ACTH assay was assessed against a leading non-isotopic method chosen as true reference on 118 samples and the following correlation was obtained: LIAISON ACTH = x non-isotopic ACTH method 1.32 with a correlation coefficient r = The nominal values of the LIAISON ACTH calibrators have been assigned to reflect the accuracy data. In order to reference calibrators values to the international standard WHO 74/555, a multiplying factor of 1.85 has to be used. Non isotopic ACTH method, Rev. E