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Procedure:
Tobramycin
OSR4S229
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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CLSIOSR4S229.02
Page 1 of 16
Procedure:
Tobramycin
OSR4S229
PRINCIPLE:
Tobramycin is an aminoglycoside antibiotic used to treat infections caused by
many different bacteria.
Monitoring serum tobramycin concentrations, along with careful clinical
assessment, is the most effective means of ensuring adequate therapy for
several reasons:

Tobramycin concentration in serum correlates better with antibacterial
activity than does dosage.1,2

A standard dose of tobramycin does not always yield a predictable serum
level because the drug's concentration also depends on the patient’s
volume of distribution and on drug elimination. These factors are
influenced by the mode of administration, the volume of extra-cellular
fluid, renal function, and physiological changes during therapy. 1,2

Tobramycin is safe and effective only in a narrow range of concentrations
for a given indication.

Exposure to high tobramycin concentrations for a prolonged period may
cause renal impairment or ototoxicity.2,3

Patients with impaired renal function should be monitored closely while
on tobramycin therapy because nephrotoxicity caused by tobramycin may
be difficult to distinguish from the symptoms of underlying renal disease.
In addition, patients with compromised renal function eliminate
tobramycin more slowly than patients with normal renal function.2,3
The methods historically used to monitor serum tobramycin concentrations
include immunoassays, and chromatographic assays.1,4
INTENDED USE:
The Emit 2000 Tobramycin Assay is intended for use in the quantitative
analysis of tobramycin in human serum or plasma. This Emit 2000 Assay is
packaged specifically for use on multiple Beckman Coulter AU analyzers.
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CLSIOSR4S229.02
Page 2 of 16
Procedure:
Tobramycin
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METHODOLOGY
The Emit 2000 Tobramycin Assay is a homogeneous enzyme immunoassay
technique used for the analysis of tobramycin in human serum or plasma.5
This 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 can be
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. 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.1,2
SAMPLE COLLECTION TIME:
For patients on a regimen of divided daily tobramycin doses, collect a peak
sample 30 - 60 minutes after intravenous infusion or 60 - 90 minutes after
intramuscular injection. Collect trough samples just before the next
scheduled dose.1-3 When adjusting dosage, measure peak and trough
levels over the same dosing interval to obtain accurate estimates of halflife and clearance.
For patients on a single daily dosage regimen, either collect a trough
sample 3 - 5 hours before the next scheduled dose or, if using a published
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CLSIOSR4S229.02
Page 3 of 16
Procedure:
Tobramycin
OSR4S229
nomogram to determine the proper dosing interval, collect a sample 7 - 14
hours after dosing.6
Additional instructions for patient preparation as designated by this laboratory:
TYPE:
Serum or plasma is the recommended specimen. Whole blood cannot be
used. The anticoagulants EDTA, heparin, citrate, and oxalate/fluoride
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:
According to the CAP Patient Preparation & Specimen Handling Fascicle
IV: Therapeutic Drug Monitoring/Toxicology, samples are generally
considered stable for 7 day when stored refrigerated at 2 - 8C and stable
for 1 month when stored frozen < -20C.
Preferably, blood specimens should be separated and tested immediately
after collection, or separated specimens should be frozen. Thaw frozen
specimens and test them immediately.
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CLSIOSR4S229.02
Page 4 of 16
Procedure:
Tobramycin
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Concentrations of beta-lactam antibiotics (penicillins and cephalosporins)
at therapeutic levels may inactivate tobramycin in vivo and in vitro.2,3
Analyze tobramycin specimens containing a beta-lactam antibiotic
immediately upon receipt or store them frozen (< -20C) to prevent in vitro
inactivation and low quantitation, or treat them with beta-lactamase.1.2
Samples that contain particulate matter, fibrous material, 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.7,8
Additional handling conditions as designated by this laboratory:
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CLSIOSR4S229.02
Page 5 of 16
Procedure:
Tobramycin
OSR4S229
EQUIPMENT AND MATERIALS:
EQUIPMENT:
Beckman Coulter AU400/AU400e, AU480, AU600, AU640/AU640e,
AU680, AU2700, AU5400 and AU5800 analyzers.
MATERIALS:
Emit 2000 Tobramycin Assay
Enzyme Reagent 1 - tobramycin labeled with bacterial G6PDH, Tris
buffer, bovine serum albumin, 0.09% sodium azide, stabilizers, and
preservatives.
Antibody/Substrate Reagent 2 - sheep antibodies reactive to
tobramycin, G6P, NAD, bovine serum albumin, 0.09% sodium azide,
stabilizers, and preservatives.
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 Tobramycin Calibrators
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(Cat No. 4S109)
CLSIOSR4S229.02
Page 6 of 16
Procedure:
Tobramycin
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The Emit Tobramycin Calibrators contain the following stated
tobramycin concentrations: 0 g/mL, 0.6 g/mL, 2.0 g/mL, 4.0 g/mL,
6.0 g/mL, 10 g/mL. The calibrators also contain Tris buffer, bovine
serum albumin, preservatives, and 0.09% sodium azide. Source
material from which the calibrators were derived is not biohazardous.
Storage location of the calibrator in this laboratory:
Preparation
The Emit 2000 Tobramycin Calibrators are provided ready to use and
may be used directly from the refrigerator. No reconstitution is
necessary.
Emit 2000 Tobramycin Reagents are provided ready to use; no
preparation is necessary. 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 Tobramycin Assay and Calibrators are for in vitro
diagnostic use.
2. Reagent 1 contains non-sterile mouse antibodies. Reagent 2
contains non-sterile sheep antibodies. Reagent and calibrators
contain non-sterile bovine serum albumin.
3. 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 to local
regulations.
4. Do not use the reagent kit or calibrators after the expiration date.
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CLSIOSR4S229.02
Page 7 of 16
Procedure:
Tobramycin
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5. Reagents and calibrators contain materials that may cause
sensitivity on contact with skin.
6. No known test method can offer complete assurance that products
derived from human blood are pathogen-free. Handle all materials
of human origin as though they were potentially infectious. If
exposed to solution containing materials of human origin, the user
should follow recommendations of the U.S. Occupational Safety and
Health Administration.7,8
Storage and Stability:
Unopened Emit® 2000 Tobramycin reagents are stable until the
expiration date printed on the label if stored upright and at 2 - 8C.
Refer to Assay Methodology Sheets for additional on-board stability
information. When not in use, store reagents at 2 - 8C, upright, and
with the screw caps tightly closed.
The Emit® 2000 Tobramycin calibrators should always be stored at 2 8C when not in use. Store upright. When stored as directed the
calibrators are stable until the expiration date printed on the vial
labels.
Do not freeze the reagents or calibrators or expose them to
temperatures above 32C.
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:
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CLSIOSR4S229.02
Page 8 of 16
Procedure:
Tobramycin
OSR4S229
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 discontinuation 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
Precision was determined by assaying two replicates each of in-house trilevel controls for 20 days with 2 runs per day. Precision was calculated
according to Clinical and Laboratory Standards Institute (CLSI EP5-A).
Within-Run Precision
Total Precision
Level 1
Level 2
Level 3
Level 1
Level 2
Level 3
Mean
(μg/mL)
1.5
3.8
6.6
1.5
3.8
6.6
SD
0.06
0.12
0.18
0.10
0.15
0.22
CV %
3.6
3.1
2.7
6.8
3.9
3.3
COMPARISON
Samples from patients were analyzed on the Syva® -30R Biochemical
System and the AU600. Results are shown in the following table.
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CLSIOSR4S229.02
Page 9 of 16
Procedure:
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Slope
1.039
Intercept (g/mL)
-0.139
Mean (g/mL)
Syva® -30R
AU600
Correlation Coefficient
Number of Samples
Range (g/mL)
2.85
2.82
0.996
47
0.47 – 8.75
CALIBRATION:
Perform a multi-point calibration (5AB) using a water blank (blue rack) and
the Emit® 2000 Tobramycin Calibrators: 0.6, 2.0, 4.0, 6.0, 10. 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 calibration stability to be at least 14 days.
Recalibrate as indicated by control results or whenever a new lot of
reagents is used.
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.
QUALITY CONTROL:
During operation of the Beckman Coulter AU analyzer at least one level of
control material should be tested every 8 hours. Alternate the control levels
tested and ensure that a minimum of 2 controls is assayed in every 24 hour
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CLSIOSR4S229.02
Page 10 of 16
Procedure:
Tobramycin
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period. Controls should be performed after calibration, with each new set of
reagent with the same lot number, 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 g/mL to mol/L tobramycin, multiply by 2.14.
REPORTING RESULTS:
REFERENCE RANGES:
Although optimum concentrations vary according to the indication, peak
tobramycin concentrations of 4.0 – 8.0 g/mL (8.6 – 17 mol/L) have been
reported to effectively control serious infection by organisms susceptible to
tobramycin in patients on divided daily dosages.2-4 Peak concentrations of
5.0 – 10.0 g/mL (11 – 21 mol/L) have been reported to effectively control
life-threatening infection by organisms susceptible to tobramycin.1
In patients on divided daily dosage regimens, reports show that trough
tobramycin concentrations of 1.0 - 2.0 g/mL (2.1 – 4.3 mol/L) usually
ensure that the concentration is above the minimum inhibitory
concentrations of most tobramycin-sensitive pathogens and that the drug
elimination is adequate4. Further, prolonged trough concentrations above
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CLSIOSR4S229.02
Page 11 of 16
Procedure:
Tobramycin
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2.0 g/mL (4.3 mol/L) are often associated with renal impairment and
ototoxicity. 1-3 Prolonged peak concentrations above 12 g/mL (26 mol/L)
are often associated with ototoxicity when patients are on divided daily
dosing regimens.1
In patients on single dosage regimens, reports show that trough
concentrations less than 1.0 g/mL (2.1 mol/L) allow adequate clearance
of tobramycin before the next dose5. Results from samples collected 7 – 14
hours after dosing can be plotted on a published nomogram to determine
the proper dosing interval. 6
For effective treatment, some patients may require serum levels outside
these ranges. Therefore, the expected ranges are 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.
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 g/mL at 37°C.
Interpretation of Results
The concentration of tobramycin in serum or plasma depends on the
time of the last drug dose; mode of administration; concomitant drug
therapy; sample condition; time of sample collection; and individual
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CLSIOSR4S229.02
Page 12 of 16
Procedure:
Tobramycin
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variations in absorption, distribution, biotransformation, and
excretion. These parameters must be considered when interpreting
results.1,2,4
The factors that can influence the relationship between tobramycin
serum or plasma concentrations and clinical response include renal
function, the susceptibility of the infecting organism to tobramycin, the
type and severity of infection, general state of health and use of other
drugs.1,2,4
Additional reporting information as designated by this laboratory:
LIMITATIONS:
The Emit® 2000 Tobramycin Assay accurately quantitates tobramycin
concentrations in human serum or plasma containing 0.6 - 10 g/mL
(1.3 - 21 mol/L) tobramycin.
To estimate tobramycin concentrations above the assay range, patient
samples containing more than 10 g/mL (21 mol/L) tobramycin may be
diluted with one or two parts distilled or deionized water or Emit® 2000
Tobramycin 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.
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CLSIOSR4S229.02
Page 13 of 16
Procedure:
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INTERFERING SUBSTANCES
Patient samples containing amikacin or kanamycin cannot be reliably
quantitated by this assay.
No clinically significant interference has been found in samples to which
800 mg/dL hemoglobin, 750 mg/dL triglycerides, or 30 mg/dL free bilirubin
were added to simulate hemolytic, lipemic, or icteric samples.
SENSITIVITY
The sensitivity level of the Emit® 2000 Tobramycin Assay is 0.45 g/mL.
This level represents to lowest concentration of tobramycin that can be
distinguished from 0 g/mL with a confidence level of 5%.
SPECIFICITY
The Emit 2000 Tobramycin Assay measures the total (protein-bound plus
unbound) tobramycin concentration in serum or plasma. Compounds
whose chemical structure or concurrent therapeutic use would suggest
possible cross-reactivity have been tested.
Amikacin cross-reacts with this assay. Kanamycin cross-reacts
significantly also. However, aminoglycosides are not generally coadministered in clinical practice although more than one aminoglycoside
may be present when switching from one treatment to another. Samples
that contain tobramycin in combination with either amikacin or
kanamycin cannot be reliable quantitated by this assay.
The compounds listed in the following table do not interfere with the
Emit 2000 Tobramycin Assay when tested in the presence of 4.0 g/mL
tobramycin. Levels tested were at or above maximum physiological or
pharmacological concentrations.
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CLSIOSR4S229.02
Page 14 of 16
Procedure:
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Compound
Concentration Tested
(g/mL)
Carbenicillin
1000
Cephalothin
1000
Chloramphenicol
1000
Clindamycin
1000
Erythromycin
1000
Neomycin
100
Netilmicin
100
Penicillin G
1000*
Sisomicin
100
Streptomycin
100
Sulphamethoxazole
60
Tetracycline
1000
Trimethoprim
25
Vancomycin
200
* Approximately equivalent to 1666 units/mL penicillin G.
REFERENCES:
1. Matthews SJ: Aminoglycosides, in Schumacher GE (ed): Therapeutic Drug
Monitoring. Norwalk, CT: Appleton and Lange; 1995:237-265.
2. Chambers HF, Sande MA: Antimicrobial agents: The Aminoglycosides, in
Hardman JG, Limbird LE (eds): Goodman and Gillman’s The
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CLSIOSR4S229.02
Page 15 of 16
Procedure:
Tobramycin
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Pharmacologic Bases of Therapeutics. 9th Ed. New York, NY: McGrawHill; 1996:1103-1121.
3. Physician’s Desk Reference. 54th Ed. Montvale, NJ: Medical Economics
Data Productions; 2000:1628-1631.
4. Moyer TP: Therapeutic drug monitoring, in Burtis CA, Ashwood ER (eds):
Tietz Textbook of Clinical Chemistry. 3rd Ed. WB Saunders; 1999:886-905.
5. Hsu P, Ernst R, Levy M: Emit® 2000 tobramycin and vancomycin assays
[abstract]. Clin Chem 2000; 46(suppl 6):page A195. Abstract 762.
6. Anaizi N: Once-daily dosing of aminoglycosides: a consensus document.
Int J Clin Pharmacol Ther. 1997 Jun;5(6):223-226.
7. Occupational exposure to bloodborne pathogens (29 CFR 1910.1030).
Federal Register. December 06, 1991;56:64004; amended April 13,
1992;57:12717; July 01, 1992;57:29206; February 13, 1996;61:5507.
8. World Health Organization. Laboratory Biosafety Manual. 2nd Ed.
Geneva: World Health Organization; 1993.
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CLSIOSR4S229.02
Page 16 of 16