Prepared By - Beckman Coulter



This procedure is valid for the following chemistry analyzers:

|AU400/AU400e |AU640/AU640e |

|AU480 |AU680 |

|AU600 |AU2700/AU5400 |

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PRINCIPLE:

Digoxin increases the strength of heart muscle contractions and is useful in the treatment of congestive heart failure. Digoxin slows the electrical conduction between the atria and ventricles of the heart, which makes it useful in treating atrial fibrillation, atrial flutter, and atrial tachycardia.

Monitoring serum digoxin concentrations, along with careful clinical assessment, is the most effective means of ensuring safe and effective therapy for several reasons:1–3

• Studies have shown a relationship between serum digoxin concentrations and clinical signs of toxicity.

• Clinical manifestations of digoxin toxicity (cardiac disturbances, gastrointestinal problems, and central nervous system disorders) can mimic those of disease processes.

• Concomitant use of other drugs, particularly quinidine, can markedly alter serum digoxin concentrations.

• Digoxin has a narrow range of safe and effective concentrations in serum. Although the therapeutic and toxic concentrations overlap, measurement of digoxin levels helps to maintain effective concentrations and to diagnose and prevent overdosage.

Methods historically used to monitor serum digoxin concentrations include radioimmunoassay, fluorescence polarization immunoassay, and enzyme immunoassay.1,2,4 Because the Emit( 2000 homogeneous enzyme immunoassay uses an enzyme label, it eliminates some difficulties that have been associated with radioimmunoassay techniques.4

INTENDED USE:

The Emit® 2000 Digoxin Assay is intended for use in the quantitative analysis of digoxin in human serum or plasma. The Emit® 2000 assays are designed for use on multiple Beckman Coulter AU analyzers.

METHODOLOGY

The enzyme in the Emit( 2000 Digoxin Assay is manufactured using recombinant DNA technology. The assay is a homogeneous enzyme immunoassay technique used for the analysis of digoxin and its active metabolites in serum or plasma5. The assay is based on competition between drug in the sample and drug labeled with recombinant glucose-6-phosphate dehydrogenase (rG6PDH) for antibody binding sites. Enzyme activity decreases upon binding to the antibody, so the drug concentration in the sample can be measured in terms of enzyme activity. Active enzyme converts oxidized nicotinamide adenine dinucleotide (NAD) to NADH, resulting in an absorbance change that is measured spectrophotometrically. Endogenous serum G6PDH does not interfere because the coenzyme functions only with the recombinant variant of the bacterial (Leuconostoc mesenteroides) enzyme employed in the assay.

SPECIMEN:

Patient / Sample Preparation:

No special preparation for the patient is required. The patient’s clinical condition and dosage regimen may influence the sample collection time.

Pharmacokinetic factors influence the correct time of sample collection after the last drug dose. These factors include dosage form, mode of administration, concomitant drug therapy, and biological variations affecting drug disposition.1-3

Sample Collection Time:

For reliable interpretation of results, collect samples either after the drug’s distribution phase or immediately before the next oral dose (at least 6 hours after administration). Samples drawn before the drug has completed its distribution phase will not accurately reflect the level of drug in the myocardium. These samples cannot be used to evaluate cardiac response, as serum levels do not represent tissue levels until at least 6 hours after oral dose or 4 hours after an intravenous dose. To evaluate maintenance doses, collect samples when digoxin levels are at steady state—the time to reach steady state is normally from three to five elimination half-lives but may be prolonged in patients with impaired renal function.1,3

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Type:

Serum or plasma is the recommended specimen. Use of fresh samples is preferred. Whole blood cannot be used. The anticoagulants heparin, oxalate, and EDTA have been tested in plasma samples containing 1.0 ng/mL digoxin. No discernible difference was observed in digoxin recovery from plasma samples as compared with serum samples.

Severely lipemic and hemolyzed samples should be avoided as they may cause poor reproducibility and questionable quantitation.

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Handling Conditions:

If samples are to be tested within 8 hours of collection, they may be stored at a room temperature (15-25°C). For transporting, maintain the sample temperature at 2-8°C. Samples can be stored refrigerated at 2-8°C for up to 7 days or stored frozen ((-20°C) for up to 6 months. Repeated freeze-thaw cycles should be avoided.

Samples that contain particulate matter, fibrous material, or gel-like masses; appear unusual; or are frozen require preparation. Use the following instructions to prepare such samples:

1. If sample is frozen, thaw at a room temperature of 15-25°C.

2. Vigorously mix sample in a vortex for at least 30 seconds.

3. Centrifuge sample at > 2000 rpm for 15 minutes.

4. Collect a specimen from the middle portion of the sample. Avoid collecting lipids from the top portion or particulate matter from the bottom portion.

Human serum or plasma samples should be handled and disposed of as if they were potentially infectious.

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EQUIPMENT AND MATERIALS:

Equipment:

Beckman Coulter AU400/AU400e, AU480, AU600, AU640/AU640e, AU680, AU2700, and AU5400 analyzers.

Materials:

Emit( 2000 Digoxin Assay

Antibody/Substrate Reagent 1 — rabbit antibodies reactive to digoxin, glucose-6-phosphate, nicotinamide adenine dinucleotide, acidic amphoteric dipeptide buffer, preservatives, and stabilizers.

Enzyme Reagent 2 — digoxin labeled with recombinant glucose-6-phosphate dehydrogenase, HEPES/Tris buffer, preservatives, and stabilizers.

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Test tubes 12 -16 mm in diameter or sample cups (Cat No. AU1063).

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Emit( 2000 Digoxin Calibrators (Cat No. 4H209)

The Emit( 2000 Digoxin Calibrators contain the following stated digoxin concentrations: 0 ng/mL, 0.5 ng/mL, 1.0 ng/mL, 2.0 ng/mL, 3.0 ng/mL, 5.0 ng/mL. The calibrator kit is sold separately.

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Preparation

The Emit( 2000 Digoxin Assay reagents and calibrators are packaged in a ready to use liquid form and may be used directly from the refrigerator. Close the calibrator vials when not in use. Caps must always be replaced on the original containers.

Note: Reagents 1 and 2 are provided as a matched set. They should not be interchanged with components of kits with different lot numbers.

Precautions:

1. The Emit( 2000 Digoxin Assay and Calibrators are for in vitro diagnostic use.

2. Reagent 1 contains non-sterile rabbit antibodies. Reagent 1 and 2 contain bovine serum albumin.

3. Do not use the reagent kit or calibrators after the expiration date.

4. Reagents and calibrators contain a preservative that may cause sensitivity on contact with skin.

Storage Requirements:

Any reagents not loaded in the reagent refrigerator on the analyzer or any calibrators not in use should be store at 2-8°C (36-46°F), upright, and with caps tightly closed. Do not freeze reagents or calibrators or expose them to temperatures above 32°C.

Unopened reagents and calibrators are stable until the expiration date printed on the label if stored as directed. Refer to Assay Methodology Sheets for additional on-board stability information.

Improper storage of reagents or calibrators can affect assay performance. Stability depends on handling reagents or calibrators as directed.

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Indications of Deterioration:

Discoloration (especially yellowing) of the reagent or calibrators, visible signs of microbial growth, turbidity, or precipitation in reagent or calibrators may indicate degradation and warrant discontinuance of use.

PERFORMANCE PARAMETERS:

The following performance characteristics represent total system performance and should not be interpreted to refer only to reagents. Studies were performed on the Beckman Coulter AU analyzer series. Results may vary due to analyzer-to-analyzer differences.

Precision

Within run precision was calculated by running twenty replicates of each level of a tri-level control. Total precision was calculated according to Clinical and Laboratory Standards Institute (CLSI EP5-A) using data collected from controls run in duplicate twice daily over twenty days (N=80).

| |Within Run Precision |Total Precision |

| |Level 1 |Level 2 |Level 3 |Level 1 |Level 2 |Level 3 |

|Mean (ng/mL) |0.7 |1.8 |2.9 |0.7 |1.8 |2.9 |

|CV/% |8.0 |6.9 |6.7 |10.4 |9.0 |9.3 |

Comparison

Samples were analyzed using the Emit( 2000 Digoxin Assay on the

SYVA(-30R and the AU600 analyzer. A summary of the results is below

|Slope |1.05 |

|Intercept (ng/mL) |-0.05 |

|Mean (ng/mL) | |

|SYVA(-30R |1.54 |

|AU600 |1.57 |

|Correlation Coefficient |0.998 |

|Number of Samples |50 |

CALIBRATION:

Perform a multi-point calibration (5AB) using a water blank (blue rack) and the Emit® 2000 Digoxin Calibrators: 0.5, 1.0, 2.0, 3.0, 5.0. Calibrator concentration is traceable to USP. Calibration parameters are set to prepare the calibration curve. Refer to analyzer User’s Guide or Analyzer Specific Protocol sheets for analyzer settings.

Calibration Stability

Studies have shown the median calibration stability to be at least 14 days. Recalibrate as indicated by control results or with a new lot of reagent. Calibration stability may vary from laboratory to laboratory depending on the following: handling of reagents, maintenance of analyzer, adherence to operating procedures, establishment of control limits, and verification of calibration.

Note: When using a new set of reagents with the same lot number, validate the system by assaying controls.

QUALITY CONTROL:

During operation of the Beckman Coulter AU analyzer at least one level of control material should be tested every 8 hours. Alternating the control levels tested and ensuring that a minimum of 2 controls is assayed in every 24 hour period. Controls should be performed after calibration, with each new set of reagent with the same lot number, with each new lot, and after specific maintenance or troubleshooting steps described in the appropriate User’s Guide. Quality control testing should be performed in accordance with regulatory requirements and individual laboratory’s standard procedures. If more frequent verification of test results is required by the operating procedures within your laboratory, those requirements should be met.

PARAMETERS:

A complete list of test parameters and operating procedures can be found in the appropriate User’s Guide and at .

CALCULATIONS:

Results are calculated automatically by the analyzer. No additional manipulation of data is required.

This assay uses Math Model No. 1.

To convert from ng/mL to nmol/L digoxin, multiply by 1.28.

REPORTING RESULTS:

Reference Ranges:

The therapeutic range of digoxin serum concentrations is 0.8 - 2.0 ng/mL (1.02-2.56 nmol/L) which includes effective serum concentrations for a wide range of patient populations.8,9

Lower concentrations of 0.5 – 1.2 ng/mL (0.64 – 1.54 nmol/L) have been found to be more appropriate in certain populations such as chronic heart failure patients.8,9

In general, toxicity is associated with levels above 2.0 ng/mL (2.6 nmol/L), but may occur with lower digoxin levels. Significant overlap of toxic and on toxic values has been reported. Consequently, analysis of serum concentrations alone is not sufficient for optimization of digoxin therapy. Additional factors such as age, thyroid condition, electrolyte balance, hepatic and renal functions, and other clinical symptoms must be considered.

Each laboratory should determine the appropriateness of this range for the diagnostic evaluation of patient results. For effective treatment, some patients may require serum levels outside this range. Therefore, the expected range is provided only as a guide, and individual patient results should be interpreted in light of other clinical signs and symptoms.

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Procedures for Abnormal Results

The laboratory must define procedures to be used in reporting high concentration (toxic) results to the patient’s physician.

Abnormal results are flagged by the listed analyzers according to the normal values entered by the user into the analyzer parameters.

Reporting Format:

Results are automatically printed out for each sample in ng/mL at 370C.

Interpretation of Results

The factors that can influence the relationship between the measured digoxin serum or plasma concentrations and clinical response include kidney function, age, electrolyte balance, tissue oxygenation, thyroid status, autonomic nervous system tone, type and severity of heart disease, and coadministered drugs.1, 3

The concentration of digoxin in serum or plasma depends on the time of the last drug dose; dosage form; mode of administration; concomitant drug therapy; sample condition; time of sample collection; and individual variations in absorption, distribution, biotransformation, and excretion. These parameters must be considered when interpreting results.1,3

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LIMITATIONS:

The Emit® 2000 Digoxin Assay accurately quantitates digoxin concentrations in human serum or plasma containing 0.2 – 5.0 ng/mL (0.3 – 6.4 nmol/L) digoxin.

To estimate digoxin concentrations above the assay range, patient samples containing more than 5.0 ng/mL (6.4 nmol/L) digoxin may be diluted with one part Emit® 2000 Digoxin Calibrator 0. After diluting the sample, repeat the entire assay sequence and multiply the results by the dilution factor.

Adulteration of reagents, use of analyzers without appropriate capabilities, or other failure to follow instructions as set forth in this protocol or the package insert can affect performance characteristics and stated or implied labeling claims.

Interfering Substances

No clinically significant interference has been found in samples to which 800 mg/dL hemoglobin, 30 mg/dL bilirubin, or 80 mg/mL human gamma globulins were added to simulate hemolytic, icteric, or hypergammaglobulinemic samples.

Endogenous, digoxin-like immunoreactive factors (DLIF) have been detected in the serum and plasma of neonates, pregnant women, and patients in renal and hepatic failure. Several studies have established that these factors can cause falsely elevated digoxin measurements when assayed by commercially available immunoassays.6

In rare instances, individuals have antibodies that interfere with the assay by depressing its enzymatic rate. This rate depression may cause low test results.

Fab fragments of antidigoxin antibodies, found in the serum and plasma of individuals being treated for digoxin intoxication, have the potential to interfere with any immunoassay in which they are not separated from digoxin before testing.7

Sensitivity

The sensitivity level of the Emit® 2000 Digoxin Assay is 0.2 ng/mL. This level represents the lowest measurable concentration of digoxin that can be distinguished from 0 ng/mL with a confidence level of 95%.

Specificity

The Emit® 2000 Digoxin Assay measures the total (protein-bound plus unbound) digoxin concentration in serum or plasma. Compounds whose chemical structure or concurrent therapeutic use would suggest possible cross-reactivity have been tested. The compounds listed below do not interfere with the Emit® 2000 Digoxin Assay when tested in the presence of 1.0 (g/mL digoxin. Levels tested were at or above maximum physiological or pharmacological concentrations.

|Therapeutic Drugs |

| Compound |Conc. Tested |

| |((g/mL) |

|Furosemide |50 |

|Hydrochlorothiazide |100 |

|Lidocaine |100 |

|Phenytoin |100 |

|Procainamide |100 |

|Propranolol |100 |

|Quinidine |100 |

|Secobarbital |100 |

|Spironolactone |10 |

|Endogenous Substances and |Concentration Tested (µg/mL) |

|Synthetic Hormones | |

|Cholesterol |10 |

|Cortisol |10 |

|Cortisone |10 |

|Estriol |10 |

|Prednisolone |10 |

|Prednisone |10 |

|Progesterone |10 |

|Testosterone |5 |

REFERENCES:

1. Keys PW, Stafford RW. Digoxin: Therapeutic use and serum concentration monitoring. In Taylor WJ, Finn AL eds. Individualizing Drug Therapy: Practical Applications of Drug Monitoring. New York: Gross, Townsend, Frank, Inc. 1981, vol 3: pp 1–21.

2. Lewis RP. Clinical Use of Serum Digoxin Concentrations. Am J Cardiol 1992. 69:97G–107G.

3. Winter ME. Digoxin. In Koda-Kimble MA, Young LY (eds): Basic Clinical Pharmacokinetics, Ed 2. Spokane, Washington: Applied Therapeutics, Inc, 1988, pp 147–171.

4. Chard T. An Introduction to Radioimmunoassay and Related Techniques, Ed 4. Laboratory Techniques in Biochemistry and Molecular Biology, Burdon RH, van Knippenberg PH eds. Amsterdam: Elsevier Science Publishers, 1990, vol 6 (pt 2): pp 73–93.

5. Pincus MR, Abraham NZ Jr: Toxicology and Therapeutic Drug Monitoring. In Henry JB ed: Clinical Diagnosis and Management by Laboratory Methods, Ed 18. Philadelphia: WB Saunders Co, 1991, pp 349–384.

6. Stone JA, Soldin SJ: An update on digoxin. Clin Chem. 1989. 35(7):1326–1331.

7. Rainey PM: Effects of Digoxin Immune Fab (ovine) on Digoxin Immunoassays. Am J Clin Pathol. 1990. 92:779–786.

8. Jogestrand T, Edner M, Haverling M. Clinical value of serum digoxin assays in outpatients: Improvement by the standardization of blood sampling. Am Heart J. 1989. 117(5):1076–1083.

9. 2009 focused Update Incorporated into the ACC/AHA 2005 Guidleines for the Diagnosis and Management of Heart Failure in Adults: A Report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines: Developed in Collaboration with the International Society for Heart and Lung Transplantation, Circulation, 2009; 119:e391-e479.

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