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3
Digoxin Assay
September 2010 4H072.3D_B
See shaded sections:
Updated information from March 2008 edition.
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 plasma.5 The assay is based on
competition between drug in the sample and drug labled 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.
4
Reagents
Reagents contain the following substances:
Rabbit antibodies reactive to digoxin (0.01 µg/mL), glucose-6-phosphate (9.6 mM),
nicotinamide adenine dinucleotide (5.6 mM), bovine serum albumin, acidic amphoteric
dipeptide buffer, digoxin labeled with recombinant glucose-6-phosphate dehydrogenase
(0.34 U/mL), HEPES/Tris buffer, preservatives, and stabilizers.
Catalog
Number
Product Description OSR4H229
Emit ® 2000 Digoxin Assay
OSR4H618 R1 (Antibody/Substrate Reagent 1)
2 x 29 mL
OSR4H648 R2 (Enzyme Reagent 2)
2 x 13 mL
4H209UL
Quantity/
Volume
Emit ® 2000 Digoxin Calibrators*
1 x 5 mL,†
5 x 2 mL
*Traceable to USP. Required for calibrating the Emit ® 2000 Digoxin Assay. Sold separately.
Additional negative calibrator is provided.
†
Note: Reagents and calibrators are shipped ready to use in liquid form.
Note: Reagents 1 and 2 are provided as a matched set. They should not be interchanged with
components of kits with different lot numbers.
The Emit ® 2000 Digoxin Calibrators contain the following stated digoxin concentrations:
Calibrator
0
0.5
1
2
3
5
Digoxin (ng/mL)
0
0.5
1.0
2.0
3.0
5.0
Digoxin (nmol/L)
0
0.6
1.3
2.6
3.8
6.4
Precautions
• For in vitro diagnostic use.
• Contains nonsterile rabbit antibodies.
• Reagents 1 and 2 contain bovine serum albumin.
• Do not use the kit after the expiration date.
• Reagents and calibrators contain a preservative that may cause sensitivity on contact with skin.
• Turbid or yellow reagents may indicate contamination or degradation and must be discarded.
Preparation of Reagents
The Emit® 2000 Digoxin Assay reagents are provided ready to use; no preparation is necessary.
Storage of Assay Components
• Improper storage of reagents can affect assay performance.
• When not in use, store reagents upright at 2–8°C and with screw caps tightly closed.
• Unopened reagents are stable until the expiration date printed on the label if stored upright at
2–8°C (36–46°F).
• Do not freeze reagents or expose them to temperatures above 32°C.
5
Specimen Collection and Preparation
The Emit ® 2000 Digoxin Assay is a homogeneous enzyme immunoassay intended for use in
the quantitative analysis of digoxin in human serum or plasma. These reagents are packaged
specifically for use on a variety of AU® Clinical Chemistry Systems.
• Each assay requires serum or plasma. 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, ie, the concentrations of the plasma samples agreed with the
concentrations of the serum samples within the precision limits of the assay at the 1.0 ng/mL
level.
2
• Sample volume is instrument-dependent. Refer to the appropriate User’s Guide or Application
Sheet for volumes.
1
Intended Use
Summary
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,
fluoresence 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
• Use fresh samples. If samples are to be tested within 8 hours of collection, they may be
stored at room temperature (20–25°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 the sample is frozen, thaw at room temperature (20–25°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.
• Pharmacokinetic factors influence the correct time of sample collection after the last drug
dose; such factors include dosage form, mode of administration, concomitant drug therapy,
and biological variations affecting drug disposition.1–3
• Human serum or plasma samples should be handled and disposed of as if they were
potentially infectious.
• 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
because 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
6
Procedure
7
Materials Provided
Emit ® 2000 Digoxin Assay
Reagent 1
Reagent 2
LIMITATIONS OF THE PROCEDURE
• Severely lipemic and hemolyzed samples should be avoided as they may cause poor
reproducibility and questionable quantitation.
• 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
Materials Required But Not Provided
Emit ® 2000 Digoxin Calibrators
Multi-level commercial controls
• 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
Refer to the instrument User’s Guide for appropriate instrument checks and maintenance
instructions.
Calibration
Calibrate at least once every six months according to the instrument settings or instructions
stated in the Application Sheet. Recalibrate whenever a new lot of reagents is used or as
indicated by control results (See Quality Control, below). If a new set of reagents with the same
lot number is used, validate the system by assaying controls. Once calibration has been verified,
run patient samples.
8
EXPECTED VALUES
The Emit ® 2000 Digoxin Assay measures digoxin concentrations in human serum or
plasma containing 0.2–5.0 ng/mL (0.3–6.4 nmol/L) digoxin. The therapeutic range of
0.8–2.0 ng/mL [1.02–2.56 nmol/L] includes effective serum concentrations for a wide range of
patient populations, although 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
Digoxin toxicity is commonly associated with serum levels > 2.0 ng/mL (2.6 nmol/L) but may
occur with lower digoxin levels. Significant overlap of toxic and nontoxic 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.
Quality Control
Each laboratory must establish and follow its own quality control procedures; however,
Siemens Healthcare Diagnostics recommends that you at least perform quality control
procedures as described below.
Temporary Control Limits. When establishing control limits for the first time, assay 10 replicates
each of multi-level (2 or more) controls to determine a mean control concentration for each
control level. Locate each mean control concentration and its corresponding precision limit
in Table 1. Ensure that the coefficient of variation (CV) of each mean control concentration is
within its precision limit. If any control is not within its precision limit, recalibrate and repeat the
10 replicates at each level. If any level fails to meet the criterion a second time, call for technical
assistance.
Each laboratory should determine the appropriateness of this range for the diagnostic evaluation
of patient results.
Table 1 — Within-Run Precision Limits
9
Mean Control Concentration
(ng/mL)
Precision Limit
(%CV)
0.50–0.80
<15
0.81–1.20
<12
>1.2
<10
Note: To convert from ng/mL to nmol/L digoxin, multiply by 1.28.
The information presented in this section is based on Emit ® 2000 Digoxin Assay studies
performed on the AU400®/AU600® Clinical Chemistry System. Refer to the Application Sheets
for other AU Clinical Chemistry Systems and for additional information. Results may vary due
to analyzer-to-analyzer differences. The following performance characteristics represent total
system performance and should not be interpreted to pertain only to reagents.
Endogenous Substances
No clinically significant interference has been found in samples to which 800 mg/dL hemoglobin,
30 mg/dL bilirubin, or 80 mg/mL gamma globulin were added to simulate hemolytic, icteric, or
hypergammaglobulinemic samples.
Use the mean control concentrations to determine temporary control limits as listed in Table 2.
Table 2 — Control Limits
Mean Control Concentration
(ng/mL)
0.50–0.80
0.81–1.20
>1.20
SPECIFIC PERFORMANCE CHARACTERISTICS
Control
Limits
–x ± 25%
Precision
Within-run precision was determined by assaying 2 replicates of each level of a tri-level control
twice a day for twenty days (N=80). Total precision data were also calculated from these data.
Tables 1 and 2 summarize the findings.
–x ± 25%
–x ± 15%
Table 1 — Within-Run Precision
Level 1
Level 2
Level 3
Mean (ng/mL) 0.7 1.8 2.9
Use these temporary limits for at least 30 days—a minimum of 20 determinations at each level
must be completed before establishing permanent control limits.
%CV 8.0 6.9 6.7
Permanent Control Limits. After 30 days (and a minimum of 20 determinations), recalculate
the mean control concentrations, including all data that are within ± 3 standard deviations.
Determine the permanent control limits by referring to Table 2. Establish new permanent control
limits whenever a new lot of controls is used.
Table 2 — Total Precision
Level 1
Level 2
Level 3
Mean (ng/mL) 0.7 1.8 2.9
Daily Quality Control. Assay at least one control every eight hours, alternating the control levels
tested. Ensure that a minimum of two controls is assayed in every 24-hour period and that
control results fall within your acceptable limits. If any control result is not within the established
control limits, despite repeat testing or recalibration, call for technical assistance. Once you have
validated the calibration curve, run patient samples.
%CV 10.4 9.0 9.3
Comparative Analysis
In this study, patient samples were analyzed on the SYVA®-30R Biochemical System and on the
AU600 Clinical Chemistry System. Table 3 summarizes the results.
Diluting High Concentration Samples
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. Some analyzers dilute and retest high concentration samples automatically. Refer
to the appropriate analyzer User’s Guide or Application Sheet for additional instructions.
Table 3 — Comparative Analysis Results
Slope
Intercept (ng/mL)
-0.05
Mean (ng/mL)
Evaluation and Interpretation of Results
• This assay uses Math Model No. 1.
• Results are automatically calculated; no additional manipulation of the data is required.
• 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 coadmininistered drugs.1,3
1.05
SYVA®-30R
1.54
AU600
1.57
Correlation Coefficient
Number
50
0.998
• 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
2
4H072.3D_B
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.
Symbols Key
Do not reuse
The compounds listed in Table 4 do not interfere with the Emit® 2000 Digoxin Assay when tested
in the presence of 1.0 ng/mL digoxin. Levels tested were at or above maximum physiological or
pharmacological concentrations.
Use By
Batch Code
Table 4 — Compounds That Do Not Interfere
Compound
Concentration Tested
(µg/mL)
Therapeutic Drugs Furosemide
Compound
Caution, consult accompanying
documents
Endogenous Substances
and Synthetic Hormones
Cholesterol
10
Hydrochlorothiazide
100
Cortisol
10
Lidocaine
100
Cortisone
10
Phenytoin
100
Estriol
10
Procainamide
100
Prednisolone
10
Propranolol
100
Prednisone
10
Quinidine
100
Progesterone
10
Secobarbital
100
Testosterone
5
Spironolactone
Catalogue Number
Concentration Tested
(µg/mL)
50
Manufacturer
Authorized Representative in the
European Community
Contains sufficient for <n> tests
IVD
In Vitro Diagnostic Medical Device
Temperature Limitation
Consult Instructions for Use
Non-sterile
10
Sensitivity
CE Mark
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%.
Contents
Calibration Stability
Studies have shown calibration stability to be at least 14 days. Calibration stability may vary
from laboratory to laboratory depending on the following: handling of reagents, maintenance of
instruments, adherence to operating procedures, establishment of control limits, and verification
of calibration.
Level
Reconstitution Volume
2010-07_BC
10 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 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. ESC Guidelines for diagnosis and treatment of patients with heart failure 2008. Eur. J Heart
Failure 10: 933-989, 2008
9. 2009 Focused Update Incorporated into the ACC/AHA 2005 Guidelines 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.
For technical assistance:
Beckman Coulter customers contact their technical assistance center.
1-800-223-0130
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®
Revised September 2010
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4H072.3D_B