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Procedure:
Theophylline
OSR4P229
This procedure is valid for the following chemistry analyzers:

AU400/AU400e

AU640/AU640e

AU480

AU680

AU600

AU2700/AU5400
Prepared By
Date Adopted
Supersedes Procedure #
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Revision Date
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Page 1 of 14
Rev # 1, Dec 31, 10
Procedure:
Theophylline
OSR4P229
PRINCIPLE:
Theophylline is a bronchodilatory drug used in the treatment of asthma and
other airway diseases. Asthma is a breathing problem involving narrowing of
the airways causing shortness of breath, wheezing, cough and congestion.
Theophylline opens airways by relaxing the smooth muscles in the airway
walls. It can also be helpful in patients with chronic bronchitis and
emphysema when their symptoms are related to airway narrowing.
The physiological effects of the anti-asthmatic drug theophylline correlate
better with the drug's concentration in serum than with dosage. Since
serious toxic effects of theophylline are related to the serum concentration
and are not always preceded by minor adverse symptoms, serum theophylline
monitoring helps to avoid serious toxicity.1-5
When theophylline is used to treat acute symptoms, monitoring serum
concentrations allows the physician to adjust the dosage regimen to
compensate for interpatient variations in the theophylline elimination rate1.
The chronic treatment of asthma and other bronchospastic diseases also
requires individualization of the theophylline dosage to maintain serum
concentrations within the therapeutic range.2,3 A theophylline dosage
generally can be maintained without further monitoring for six months in
rapidly growing children and for twelve months in other patients. Changes
in concurrent drug therapy, variations in drug elimination, or the appearance
of side effects, uncontrolled symptoms, or altered drug clearance signal the
need for measuring the serum theophylline concentration.1,3
Methods historically used to monitor serum theophylline concentrations
include immunoassay, gas-liquid chromatography (GLC) assay and highperformance liquid chromatography (HPLC).1,5, 6
INTENDED USE:
The Emit® 2000 Theophylline Assay is intended for use in the quantitative
analysis of theophylline in human serum or plasma. The Emit® 2000 assays
are designed for use on multiple Beckman Coulter AU analyzers.
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 2 of 14
Rev # 1, Dec 31, 10
Procedure:
Theophylline
OSR4P229
METHODOLOGY:
The Emit® 2000 Theophylline Assay is a homogeneous enzyme immunoassay
technique used for the analysis of specific compounds in biological fluids.7,8
The assay is based on competition between drug in the sample and drug
labeled with the enzyme glucose-6-phosphate dehydrogenase (G6PDH) 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 bacterial (Leuconostoc
mesenteroides) enzyme employed in the assay.
SPECIMEN:
PATIENT / SAMPLE PREPARATION:
No special preparation for the patient is required. Patient intake of
caffeinated beverages does not need to be restricted. 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.1-5
SAMPLE COLLECTION TIME:
Measure the steady-state serum concentration representing the trough
level just before the next scheduled dose.1
Additional instructions for preparation as designated by this laboratory:
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All printed copies are considered to be copies of the electronic original.
Page 3 of 14
Rev # 1, Dec 31, 10
Procedure:
Theophylline
OSR4P229
TYPE:
Serum or plasma is the recommended specimen. Whole blood cannot be
used. The anticoagulants heparin, citrate, oxalate, and EDTA have been
tested and may be used with this assay.
Some sample dilution may occur when samples are collected in tubes
containing citrate anticoagulant. The amount of dilution and the possible
need to correct for it should be considered when interpreting assay results
for these samples.
Additional type conditions as designated by this laboratory:
HANDLING CONDITIONS:
Store the serum or plasma refrigerated at 2-8°C. For transporting,
maintain the sample temperature at 2-8°C. Samples can be stored
refrigerated at 2-8°C for up to one month or stored frozen (-20°C) for up
to 3 months.
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 completely at a room temperature of 1525°C.
2. Vigorously mix all samples: Vortex for at least 30 seconds.
3. Centrifuge sample at  2000 rpm for 15 minutes.
4. Collect a sample from the middle portion of the specimen. Avoid
collecting lipids from the top portion or particulate matter from the
bottom portion.
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 4 of 14
Rev # 1, Dec 31, 10
Procedure:
Theophylline
OSR4P229
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, and AU5400 analyzers.
MATERIALS:
Emit 2000 Theophylline Assay
Antibody/Substrate Reagent 1 — mouse monoclonal antibodies
reactive to theophylline, glucose-6-phosphate, nicotinamide adenine
dinucleotide, preservatives, 0.1% sodium azide, and stabilizers.
Enzyme Reagent 2 — theophylline labeled with glucose-6-phosphate
dehydrogenase, Tris buffer, preservatives, 0.1% sodium azide, and
stabilizers.
Reagent storage location in this laboratory:
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All printed copies are considered to be copies of the electronic original.
Page 5 of 14
Rev # 1, Dec 31, 10
Procedure:
Theophylline
OSR4P229
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 Theophylline Calibrator Kit
(Cat No. 4P109)
The Emit 2000 Theophylline Calibrators contain the following stated
theophylline concentrations: 0 g/mL, 2.5 g/mL, 5.0 g/mL, 10 g/mL,
20 g/mL, 40 g/mL. The calibrator kit is sold separately.
Storage location of the calibrator in this laboratory:
Preparation
The Emit 2000 Theophylline 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 Theophylline reagents and calibrators are for in
vitro diagnostic use.
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 6 of 14
Rev # 1, Dec 31, 10
Procedure:
Theophylline
OSR4P229
2. Reagent 1 contains non-sterile mouse monoclonal antibodies.
3. Do not use the reagents or calibrators after the expiration date.
4. Assay components contain sodium azide, which may react with lead
and copper plumbing to form highly explosive metal azides. If
waste is discarded down the drain, flush it with a large volume of
water to prevent azide buildup. Dispose of properly in accordance
with local regulations.
5. The reagent kit and calibrators contain streptomycin sulfate.
Please dispose of appropriately.
Storage Requirements:
Any reagents not loaded in the reagent refrigerator on the analyzer or
any calibrators not in use should be stored at 2-8°C, 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:
Indications of Deterioration:
Discoloration (especially yellowing) of the reagents or calibrators,
visible signs of microbial growth, turbidity, or precipitation in reagent
or calibrator may indicate degradation and warrant discontinuance of
use.
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 7 of 14
Rev # 1, Dec 31, 10
Procedure:
Theophylline
OSR4P229
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 (µg/mL)
5.60
14.02
28.92
5.26
13.60
28.94
CV %
2.7
3.0
3.9
6.6
7.1
11.3
COMPARISON
Samples from patients were analyzed using the Emit® 2000 Theophylline
Assay on the TDx analyzer and the AU600. The comparative analysis is
shown below.
Slope
0.96
Intercept
0.02
Mean (g/mL)
TDx
AU600
14.55
14.11
Correlation Coefficient
0.98
Number of Samples
64
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All printed copies are considered to be copies of the electronic original.
Page 8 of 14
Rev # 1, Dec 31, 10
Procedure:
Theophylline
OSR4P229
CALIBRATION:
Perform a multi-point calibration (5AB) using a water blank (blue rack) and
the Emit® 2000 Theophylline Calibrators: 2.5, 5.0, 10, 20, 40. 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 multi-level controls
should be tested a minimum of once a day. Controls should be performed
after calibration, with each new set or lot of reagent, 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.
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 9 of 14
Rev # 1, Dec 31, 10
Procedure:
Theophylline
OSR4P229
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 g/mL to mol/L theophylline, multiply by 5.55.
REPORTING RESULTS:
REFERENCE RANGES:
In most patients, theophylline serum concentrations of 10-20 g/ml (56111 mol/L) effectively suppress chronic asthmatic and other
bronchospastic symptoms.2-5 Serum concentrations of 5-10 g/ml (28-56 
mol/L) theophylline reportedly control apneic spells in neonates without
causing apparent side effects.2-4 Peak concentrations above 20 g/ml (111
mol/l) are often associated with toxicity.2-5
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:
PROCEDURES FOR ABNORMAL RESULTS
The laboratory must define procedures to be used in reporting high
concentration (toxic) results to the patient’s physician.
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 10 of 14
Rev # 1, Dec 31, 10
Procedure:
Theophylline
OSR4P229
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 for each sample in g/mL at 37C.
Interpretation of Results
The factors that can influence the relationship between the
theophylline serum or plasma concentrations and clinical response
include the type and severity of bronchial constriction, age, smoking,
diet, general state of health, and use of other drugs.2,3
The concentration of theophylline 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.2-5
Additional reporting information as designated by this laboratory:
LIMITATIONS:
The Emit® 2000 Theophylline Assay accurately quantitates theophylline
concentrations in human serum or plasma containing 2.5-40 g/mL (14-222 
mol/L) theophylline.
To estimate theophylline concentrations above the assay range, patient
samples containing more than 40 g/mL (222 mol/L) theophylline may be
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 11 of 14
Rev # 1, Dec 31, 10
Procedure:
Theophylline
OSR4P229
diluted with one or two parts distilled or deionized water or Emit 2000
Theophylline Calibrator 0. After diluting the sample, repeat the entire assay
sequence and multiply the results by the dilution factor.
Adulteration of reagents, use of analyzer 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 claims.
INTERFERING SUBSTANCES
The compound 3-isobutyl-1-methylxanthine interferes with this assay.
The compound is not a naturally occurring xanthine or known metabolite,
but some laboratories use it as an internal standard in chromatographic
procedures.9
No clinically significant interference has been found in samples to which
800 mg/dL hemoglobin, 750 mg/dL triglycerides, or 30 mg/dL bilirubin
were added to simulate hemolytic, lipemic or icteric samples.
SENSITIVITY
The sensitivity level of the Emit 2000 Theophylline Assay is 0.75 g/mL.
This level represents the lowest measurable concentration of theophylline
that can be distinguished from 0 g/mL with a confidence level of 95%.
SPECIFICITY
The Emit 2000 Theophylline Assay measures the total (protein-bound
plus unbound) theophylline concentration in serum or plasma.
Compounds whose chemical structure or concurrent therapeutic use
would suggest possible cross-reactivity have been tested.
The compounds listed in the following table do not interfere with the
Emit 2000 Theophylline Assay when tested in the presence of 10.0 g/mL
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 12 of 14
Rev # 1, Dec 31, 10
Procedure:
Theophylline
OSR4P229
theophylline. Levels tested were at or above maximum physiological or
pharmacological concentrations.
Compound
Concentration Tested
(g/mL)
Caffeine
100
8-Chlorotheophylline
25
1,3-Dimethyluric Acid
100
Dyphylline
100
Ephedrine
5
Hypoxanthine
100
1-Methyluric Acid
100
3-Methyluric Acid
200
1-Methylxanthine
30
3-Methylxanthine
100
7-Methylxanthine
100
Paraxanthine
50
Phenobarbital
100
Theobromine
100
1,3,7-Trimethyluric Acid
100
Urea
1000
Uric Acid
200
Xanthine
100
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 13 of 14
Rev # 1, Dec 31, 10
Procedure:
Theophylline
OSR4P229
REFERENCES:
1. Hendeles L, Weinberger M, Johnson G.: Monitoring Serum Theophylline
Levels. Clin Pharmacokinet 1978; 3:294-312.
2. Hendeles L, Weinberger M. Theophylline: 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; 1:31-66.
3. Hendeles L, Massanari M, Weinberger M.: Theophylline. In Middleton E
Jr, Reed CE, Ellis EF, et al. (eds): Allergy: Principles and Practice, Ed 3.
St. Louis: The CV Mosby Co. 1988; 1:pp 673-714.
4. Bierman CW, Williams PV. Therapeutic Monitoring of Theophylline:
Rationale and Current Status. Clin Pharmacokinet 1989; 17(6):377-384.
5. Glynn-Barnhart A, Hill M, Szefler SJ. Sustained release theophylline
preparations: Practical recommendations for prescribing and therapeutic
drug monitoring. Drugs. 1988; 35:711-726.
6. Rainbow SJ, Dawson CM, Tickner TR. Non-extraction HPLC method for
the simultaneous measurement of theophylline and caffeine in human
serum. Ann Clin Biochem 1989; 26:527-532.
7. 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, 349–
384.
8. Bautista J, Huster M: Syva Emit 2000 Theophylline Assay. San Jose,
CA, Syva Co.
9. Bailey DG, Davis HL, Johnson G: Improved Theophylline Serum Analysis
by an appropriate internal standard for gas chromatography. J
Chromatogr. 1976; 121:263-268.
© Beckman Coulter, Inc. 2010
All printed copies are considered to be copies of the electronic original.
Page 14 of 14
Rev # 1, Dec 31, 10