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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 Signature # of Distributed to Copies # of Distributed to © Beckman Coulter, Inc. March 2012 All printed copies are considered to be copies of the electronic original. Copies 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. © Beckman Coulter, Inc. March 2012 All printed copies are considered to be copies of the electronic original. CLSIOSR4S229.02 Page 2 of 16 Procedure: Tobramycin OSR4S229 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 © Beckman Coulter, Inc. March 2012 All printed copies are considered to be copies of the electronic original. 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 - 8C and stable for 1 month when stored frozen < -20C. Preferably, blood specimens should be separated and tested immediately after collection, or separated specimens should be frozen. Thaw frozen specimens and test them immediately. © Beckman Coulter, Inc. March 2012 All printed copies are considered to be copies of the electronic original. CLSIOSR4S229.02 Page 4 of 16 Procedure: Tobramycin OSR4S229 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 (< -20C) 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: © Beckman Coulter, Inc. March 2012 All printed copies are considered to be copies of the electronic original. 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 © Beckman Coulter, Inc. March 2012 All printed copies are considered to be copies of the electronic original. (Cat No. 4S109) CLSIOSR4S229.02 Page 6 of 16 Procedure: Tobramycin OSR4S229 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. © Beckman Coulter, Inc. March 2012 All printed copies are considered to be copies of the electronic original. CLSIOSR4S229.02 Page 7 of 16 Procedure: Tobramycin OSR4S229 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 - 8C. Refer to Assay Methodology Sheets for additional on-board stability information. When not in use, store reagents at 2 - 8C, upright, and with the screw caps tightly closed. The Emit® 2000 Tobramycin calibrators should always be stored at 2 8C 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 32C. 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. 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. © Beckman Coulter, Inc. March 2012 All printed copies are considered to be copies of the electronic original. CLSIOSR4S229.02 Page 9 of 16 Procedure: Tobramycin OSR4S229 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 © Beckman Coulter, Inc. March 2012 All printed copies are considered to be copies of the electronic original. CLSIOSR4S229.02 Page 10 of 16 Procedure: Tobramycin OSR4S229 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 © Beckman Coulter, Inc. March 2012 All printed copies are considered to be copies of the electronic original. CLSIOSR4S229.02 Page 11 of 16 Procedure: Tobramycin OSR4S229 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 © Beckman Coulter, Inc. March 2012 All printed copies are considered to be copies of the electronic original. CLSIOSR4S229.02 Page 12 of 16 Procedure: Tobramycin OSR4S229 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. © Beckman Coulter, Inc. March 2012 All printed copies are considered to be copies of the electronic original. CLSIOSR4S229.02 Page 13 of 16 Procedure: Tobramycin OSR4S229 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. © Beckman Coulter, Inc. March 2012 All printed copies are considered to be copies of the electronic original. CLSIOSR4S229.02 Page 14 of 16 Procedure: Tobramycin OSR4S229 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 © Beckman Coulter, Inc. March 2012 All printed copies are considered to be copies of the electronic original. CLSIOSR4S229.02 Page 15 of 16 Procedure: Tobramycin OSR4S229 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. © Beckman Coulter, Inc. March 2012 All printed copies are considered to be copies of the electronic original. CLSIOSR4S229.02 Page 16 of 16