Aldosterone on Liaison XL In use date: June 6, 2017

Aldosterone on Liaison XL In use date: June 6, 2017 I. PRINCIPLE Aldosterone is a steroid hormone of molecular weight 360.4 daltons, the major mineralcorticoid secreted by the adrenal cortex. The role of aldosterone in metabolism is the control of sodium and potassium and therefore it regulates fluid volume. Aldosterone acts to decrease excretion of sodium and increase the excretion of potassium at the kidney, sweat glands, and salivary glands. Aldosterone also conserves sodium in the colon. In each of these tissues aldosterone works through binding at the mineralocorticoid receptors, and primarily at the cortical collecting ducts of the kidney. Regulation of sodium and potassium balance is accomplished through a complex set of hormones acting in several feedback loops. The renin-angiotensin systems (RAS), is the most important negative feedback loop for volume regulation. The RAS operates through a long feedback loop (involving changes in fluid volume) and a short feedback loop (with direct inhibition of renin secretion by angiotensin II). The other feedback loop that acts simultaneously is the control of serum potassium. These interacting feedback loops act in concert to set aldosterone concentrations to maintain homeostasis for volume, blood pressure, and potassium in response to external stimuli. Aldosterone acts on the cortical collecting duct to increase the absorption of sodium and decreases the absorption of potassium. The resulting increase in fluid volume and blood pressure is sensed at the renin-secreting juxtaglomerular cells, which decrease the production of renin. With less renin, less angiotensin I is formed, and thereby angiotensin II levels are lowered. Lower levels of the acute stimulators of aldosterone secretion leads to decreased aldosterone synthesis and secretion. Aldosterone s role in the regulation of potassium homeostasis is also regulated primarily by a negative feedback loop. Increased potassium levels stimulate aldosterone production (aldosterone decreases reabsorption of potassium, thereby increasing potassium loss). The subsequent decrease in plasma potassium results in less potassium stimulation of the adrenal glomerulosa cells and lowers aldosterone synthesis and secretion. Renin and aldosterone measurements are used in the investigation of patients with suspected: - Primary hyperaldosteronism (PA, Conn s syndrome) is a disorder caused by excessive secretion of aldosterone by the adrenal gland, where high levels of circulating aldosterone are expected in the presence of low renin concentration or low plasmatic renin activity levels. Such inappropriately high aldosterone production (non-suppressible by sodium loading) causes cardiovascular damage, suppression of plasma renin, hypertension, sodium retention and potassium excretion that, if prolonged and severe, may lead to hypokalemia. The major causes of PA (>90% cases) are adrenal adenoma and unilateral or bilateral adrenal hyperplasia. Rare cases are due to the inherited condition of glucocorticoid-remediable aldosteronism. - Secondary hyperaldosteronism is caused by disorders that activate the renin-angiotensinaldosterone axis, resulting in excessive production of aldosterone (renovascular disease, salt depletion, potassium loading, cardiac failure with ascites, pregnancy, Bartter s syndrome). - Hypoaldosteronism is a rare condition often due to primary adrenal failure, where plasma aldosterone has low concentrations in the presence of high concentrations of plasma renin. In

hypoaldosteronism caused by low secretion of renin, low levels of plasma renin are expected in the presence of low levels of plasma aldosterone. Because many factors (age, posture, sodium and potassium balance, time of day, menstrual cycle, etc.) influence the secretion of renin and aldosterone, evaluation of these hormones should be interpreted under strictly controlled conditions. The assay of aldosterone may be carried out in serum, EDTA plasma and in 24-hour urine. From a clinical point of view it must be remembered that, while the 24-hour urinary measurement of 18-oxo-conjugate of aldosterone is an integrated reflection of the daily aldosterone secretion, the plasma values may reflect only a single point in time. Since plasma aldosterone shows typical bursts, which follow a circadian rhythm, it might not be advisable to draw conclusions from a single determination. Plasma aldosterone measurement is used for acute studies (like circadian rhythms, postural changes, acute effect of drugs), the 24-hour urinary measurement helps determine the relationship between sodium excretion and aldosterone levels and may also be used to help to rule out high aldosterone secretion as a cause of another condition. The LIAISON Aldosterone assay is a competitive assay that uses sheep monoclonal antibody for capture of the Aldosterone molecule. The principle components of the test consist of magnetic particles (solid phase) coated with anti-sheep antibody that bind sheep anti-aldosterone monoclonal antibody. An Aldosterone labeled conjugate containing an isoluminol derivative competes with Aldosterone from the calibrators, controls and patient samples. During the first incubation, sample is incubated with a specific anti-aldosterone monoclonal antibody. Following the 1st incubation the conjugate is added and competes with Aldosterone for an additional amount of time. After this second incubation the unbound material is removed with a wash cycle. The starter reagents are then added and a flash chemiluminescent reaction is initiated. The light signal is measured by a photomultiplier as relative light units (RLU) and is inversely proportional to the concentration of Aldosterone present in the calibrators, controls and patient samples. II. POLICY/SCOPE This is intended for the China Basin Chemistry section of the Clinical Laboratories and intended for testing by licensed Clinical Laboratory Scientists and Clinical Laboratory staff. III. TEST AVAILABILITY (n/a) IV. SPECIMEN REQUIRMENTS a. Preferred tube type: Serum from Red Top. b. Acceptable tube type: Serum from SST. c. Blood should be collected aseptically by venipuncture, noting the collection time and position of patient (supine or upright). d. Allow complete clot formation to take place in serum samples prior to centrifugation. e. Samples should be free of particulate matter, turbidity, lipemia, or red cells.

f. Samples should be free of bubbles. g. Specimen storage and stability: i. Serum is stable for: 1. 5 days at 2-8 C 2. 4 weeks at -20 C or colder ii. Serum is stable through 3 freeze-thaw cycles. iii. Frozen serum samples must be mixed thoroughly after thawing by low speed vortexing or by gentle inversion and centrifuged prior to use to remove red blood cells or particulate matter to ensure consistency in the results. iv. Minimum volume: 250 ul serum V. EQUIPMENT, REAGENTS, AND SUPPLIES a. LIAISON Aldosterone Reagent Kit (100 tests), Catalog # 310450 i. Storage and Stability: ii. Preparation: 1. Store reagents at 2-8 C. 2. Unopened: Stable until the expiration date on the carton. 3. Opened (2-8 C or onboard): Stable for a maximum of 6 weeks. 1. Before the seal is removed, carefully mix the magnetic particle compartment by rotating the small wheel until the color of the suspension has changed to brown. Gentle and careful side-to-side mixing may assist in the suspension of the magnetic particles (avoid foam formation). 2. Insert the reagent integral into the solid-state magnetic device located on the right-side of the instrument and let stand for at least 30 seconds. Repeat as necessary. 3. Manually stir the magnetic particles for 1 minute before placing onto the instrument. Take precaution to not create bubbles or foam. 4. Remove all seals from the reagent integral and carefully wipe the surface of each septum to remove residual liquid. Remove any bubbles that are visible on the reagent openings. If foaming of reagent occurs, place the integral on the instrument and allow the foam to dissipate before using. 5. Place the reagent integral into reagent area of the instrument with the label facing left and let it stand for 15 minutes before using. b. LIAISON Endocrinology Diluent, Catalog #319133 i. Storage and Stability: Store the diluent at 2-8 C until expiration date on bottle.

VI. WARNINGS AND PRECAUTIONS Caution: This product requires the handling of human specimens. It is recommended that all human sourced materials are considered potentially infectious and be handled in accordance with the OSHA Standard on Bloodborne Pathogens. Appropriate biosafety practices should be used for materials that contain or are suspected of containing infectious agents. VII. VIII. CALIBRATION a. LIAISON Aldosterone Calibrators Level 1 and 2 (included in reagent kit). i. Prior to use, equilibrate the calibrators to room temperature and mix thoroughly by gentle inversion. ii. Calibrator and Reagent Integral lot number are lot specific. Do not use calibrators matched with a different reagent lot in the same assay. iii. Unopened: Stable at 2-8 C until expiration date. iv. Opened: Stable for 6 weeks at 2-8 C. b. Two-point calibration is performed: i. Every 14 days. ii. When changing lot numbers of primary reagent packs. iii. When changing lot numbers of starter reagents. iv. When quality control results are repeatedly out of range. c. Refer to Liaison XL Operating Procedure for instructions on checking calibration status, performing calibration, and reviewing calibration data. QUALITY CONTROL a. BioRad Immunoassay Plus Controls Level 1, Level 2, and Level 3. i. Preparation: Reconstitute with 5.0 ml of Type 1 deionized water. ii. Stability: 7 days when stored at 2-8 C. b. See posted QC chart for acceptability limits (C-CL1, C-CL2, and C-CL3). c. Frequency: i. Analyze all levels of QC on each day that samples are analyzed. ii. Analyze all levels of QC every time a two-point calibration is performed. iii. Analyze all levels of QC on every primary reagent pack used for sample analysis. d. Refer to Liaison XL Operating Procedure for instructions on programming QC on the instrument. IX. PROCEDURE Refer to Liaison XL Operating Procedure for instructions on scheduling and loading patient samples.

X. RESULTING/REPORTABLE RANGE a. Analytical Measuring Range (AMR) i. The AMR for the Liaison Aldosterone assay is 3 to 80 ng/dl. b. Reportable Range i. The reportable range for the Liaison Aldosterone assay is 3 to 40,000 ng/dl. ii. Values below 3 ng/dl are reported as <3 ng/dl. iii. Values above 40,000 ng/dl are reported as >40,000 ng/dl. iv. Results are reported in whole numbers. v. Autoverification 1. Results between 3 and 80 ng/dl will autoverify. 2. Results <3 and >80 will be reviewed by the CLS before sending. vi. If a result is above 80 ng/dl, a manual dilution will be performed using LIAISON Endcrinology Diluent. Please see Appendix I. XI. EXPECTED VALUES ADULT ( 18 years old): Upright 8:00-10:00 am Upright 4:00-6:00 pm Supine 8:00-10:00 am < 29 ng/dl < 22 ng/dl 3-16 ng/dl Adult reference range adopted from Quest Diagnostics and verified in-house with 36 adult lab donors between the ages of 21-65 years old. Samples were collected upright between 8 am to 10 am. PEDIATRICS: 1-12 months 2-70 ng/dl 1-4 years 2-37 ng/dl 5-9 years < 10 ng/dl 10-13 years < 22 ng/dl 14-17 years < 36 ng/dl Premature Infants (31-35 weeks) < 145 ng/dl Term Infants < 218 ng/dl TANNER STAGES: MALES FEMALES Tanner Stages II-III 1-13 ng/dl 2-20 ng/dl Tanner Stages IV-V 3-14 ng/dl 4-32 ng/dl Pediatric reference range adopted from Quest Diagnostics.

XII. LIMITATIONS OF PROCEDURE a. Assay results should be utilized in conjunction with other clinical and laboratory data to assist the clinician in making individual patient management decisions. b. A skillful technique and strict adherence to the instructions are necessary to obtain reliable results. c. Grossly hemolyzed, icteric, or lipemic samples as well as samples containing particulate matter or exhibiting obvious microbial contamination are not recommended and should not be tested. d. Bacterial contamination of samples or repeated freeze-thaw cycles may affect results. e. Do not use heat inactivate serum. XIII. PERFORMANCE CHARACTERISTICS a. SPECIFICITY i. Controlled studies of potentially Cross-reactive substances in serum samples were performed on the LIAISON Aldosterone assay at the concentrations listed below. All substances showed <0.02% cross reactivity. The testing was based on the CLSI- EP7-A2. b. SENSITIVITY i. The LIAISON Aldosterone assay has an analytical sensitivity of 3 ng/dl. ii. The analytical sensitivity is defined as the concentration of Aldosterone that is two standard deviations less than the mean concentration of 20 replicate determinations of Endocrinology diluent (0 ng/dl).

c. PRECISION i. Intra-Precision study performed with BioRad Lyphochek Immunoassay Plus Controls (CL1, CL2, CL3) and Liaison XL Aldosterone Control Set (L1, L2) CV CV CV CV CV Material CL1 CL2 CL3 L1 L2 Criteria <10% <10% <10% <10% <10% Results 3% 4% 3% 2% 3% i. Inter-Precision study performed with BioRad Lyphochek Immunoassay Plus Controls (CL1, CL2, CL3) CV CV CV Material CL1 CL2 CL3 Criteria <10% <10% <10% Results 8% 9% 9% d. METHOD COMPARISON i. Laboratory performed study using xx patient samples comparing Quest Diagnostic results (LCMS) to the Diasorin Liaison XL results. Slope R value Mean Bias Criteria 0.9-1.1 > 0.95 <10% Results 0.9 1.00-4% e. INTERFERENCE i. Controlled studies of potentially interfering substances at two Aldosterone levels in serum (15 and 30 ng/dl) showed no interference in the LIAISON Aldosterone assay at the highest concentrations for each substance listed below. The testing was based on CLSI-EP7-A2.

ii. Controlled studies of potentially Cross-reactive substances in serum were performed on the LIAISON Aldosterone assay at the concentrations listed below. All substances showed <0.02% cross reactivity. The testing was based on CLSI-EP7-A2. iii. No high dose hook effect was observed for aldosterone concentrations in serum up to 1000 ng/dl.

XIV. TECHNICAL NOTES a. See Appendix I for manual dilution protocol. XV. ALTERNATE METHODS In exceptional cases, when Chemistry is unable to run the assay on the Liaison XL platform, samples may be sent to a commercial facility for testing. XVI. REFERENCES a. DiaSorin Liaison Aldosterone Assay Package Insert, REF 310450, REF 310450, US- 42980-2014-9. b. DiaSorin Liaison Endocrinology Diluent Package Insert, REF c. DiaSorin Liaison Aldosterone Control Set Package Insert, REF 310450, REF 310450, US-42498-2014-6.

Appendix I: Aldosterone Manual Dilution Protocol If the result is: >80 ng/dl Then: Make a manual 1:10 dilution by adding 50 ul of sample to 450 ul of LIAISON Endocrinology Diluent. Mix well by vortex and retest. If the diluted resulted is within AMR of 3 80 ng/dl, multiply by 10 and report the value. >800 ng/dl after 1:10 dilution Make a manual 1:100 dilution by adding 50 ul of the 1:10 dilution to 450 ul of LIAISON Endocrinology Diluent. Mix well by vortex and retest. If the diluted resulted is within AMR of 3 80 ng/dl, multiply by 100 and report the value. >8000 ng/dl after 1:100 dilution Make a manual 1:500 dilution by adding 100 ul of the 1:100 dilution to 400 ul of LIAISON Endocrinology Diluent. Mix well by vortex and retest. If the diluted resulted is within AMR of 3 80 ng/dl, multiply by 500 and report the value. >40,000 ng/dl after 1:500 dilution Report as >40,000 ng/dl