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Procedure:
Digoxin
OSR4H229
This procedure is valid for the following chemistry analyzers:

AU400/AU400e

AU640/AU640e

AU480

AU680

AU600

AU2700

AU5400

AU5800
Prepared By
Date Adopted
Supersedes Procedure #
Review Date
Revision Date
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CLSIOSR4H229.02
Page 1 of 16
Procedure:
Digoxin
OSR4H229
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.
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CLSIOSR4H229.02
Page 2 of 16
Procedure:
Digoxin
OSR4H229
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
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CLSIOSR4H229.02
Page 3 of 16
Procedure:
Digoxin
OSR4H229
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
Additional instructions for preparation as designated by this laboratory:
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.
Additional type conditions as designated by this laboratory:
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 freezethaw cycles should be avoided.
© Beckman Coulter, Inc. March 2012
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CLSIOSR4H229.02
Page 4 of 16
Procedure:
Digoxin
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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.
Additional handling conditions as designated by this laboratory:
EQUIPMENT AND MATERIALS:
EQUIPMENT:
Beckman Coulter AU400/AU400e, AU480, AU600, AU640/AU640e,
AU680, AU2700, AU5400 and AU5800 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.
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CLSIOSR4H229.02
Page 5 of 16
Procedure:
Digoxin
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Enzyme Reagent 2 — digoxin labeled with recombinant glucose-6phosphate dehydrogenase, HEPES/Tris buffer, preservatives, and
stabilizers.
Reagent storage location in this laboratory:
Test tubes 12 -16 mm in diameter or sample cups
(Cat No. AU1063).
Storage location of test tubes or sample cups in this laboratory:
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.
Storage location of the calibrator in this laboratory:
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.
© Beckman Coulter, Inc. March 2012
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CLSIOSR4H229.02
Page 6 of 16
Procedure:
Digoxin
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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.
Additional storage requirements as designated by this laboratory:
© Beckman Coulter, Inc. March 2012
All printed copies are considered to be copies of the electronic original.
CLSIOSR4H229.02
Page 7 of 16
Procedure:
Digoxin
OSR4H229
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
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All printed copies are considered to be copies of the electronic original.
CLSIOSR4H229.02
Page 8 of 16
Procedure:
Digoxin
OSR4H229
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
AU600
Correlation Coefficient
Number of Samples
1.54
1.57
0.998
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
© Beckman Coulter, Inc. March 2012
All printed copies are considered to be copies of the electronic original.
CLSIOSR4H229.02
Page 9 of 16
Procedure:
Digoxin
OSR4H229
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 www.beckmancoulter.com.
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.
© Beckman Coulter, Inc. March 2012
All printed copies are considered to be copies of the electronic original.
CLSIOSR4H229.02
Page 10 of 16
Procedure:
Digoxin
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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.
Expected reference ranges in this laboratory:
© Beckman Coulter, Inc. March 2012
All printed copies are considered to be copies of the electronic original.
CLSIOSR4H229.02
Page 11 of 16
Procedure:
Digoxin
OSR4H229
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 37C.
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
Additional reporting information as designated by this laboratory:
© Beckman Coulter, Inc. March 2012
All printed copies are considered to be copies of the electronic original.
CLSIOSR4H229.02
Page 12 of 16
Procedure:
Digoxin
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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
© Beckman Coulter, Inc. March 2012
All printed copies are considered to be copies of the electronic original.
CLSIOSR4H229.02
Page 13 of 16
Procedure:
Digoxin
OSR4H229
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
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CLSIOSR4H229.02
Page 14 of 16
Procedure:
Digoxin
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Endogenous
Substances and
Synthetic
Hormones
Concentration
Tested (µg/mL)
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,
© Beckman Coulter, Inc. March 2012
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CLSIOSR4H229.02
Page 15 of 16
Procedure:
Digoxin
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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.
© Beckman Coulter, Inc. March 2012
All printed copies are considered to be copies of the electronic original.
CLSIOSR4H229.02
Page 16 of 16