Prepared By - Beckman Coulter



This procedure is valid for the following chemistry analyzers:

|AU400/AU400e |AU640/AU640e |

|AU480 |AU680 |

|AU600 |AU2700/AU5400 |

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PRINCIPLE:

Vancomycin is an aminoglycoside antibiotic used to treat infections caused by many different bacteria.

Monitoring serum vancomycin concentrations, along with careful clinical assessment, is the most effective means of ensuring adequate therapy for several reasons:

▪ Individual patients exhibit a high degree of variability in response to a given dose of vancomycin in terms of the volume of distribution and the rate of drug clearance from plasma.1

▪ The risk of ototoxicity and nephrotoxicity from vancomycin is increased in patients with impaired renal function and in patients receiving concurrent aminoglycoside therapy.1

▪ Patients with impaired renal or hepatic function, dialysis patients, morbidly obese patients, patients receiving concurrent aminglycoside therapy, and pediatric or elderly patients should be monitored closely while on vancomycin therapy.1

The methods historically used to monitor serum vancomycin concentrations include microbiological assays, immunoassays, and chromatographic assays.1

INTENDED USE:

The Emit( 2000 Vancomycin Assay is intended for use in the quantitative analysis of vancomycin in human serum or plasma. This Emit( 2000 Assay is packaged specifically for use on multiple Beckman Coulter AU analyzers.

METHODOLOGY

The Emit( 2000 Vancomycin Assay is a homogeneous enzyme immunoassay technique used for the quantitative analysis of vancomycin in human serum or plasma2. 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 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

Sample Collection Time:

To obtain a vancomycin concentration that best represents the peak tissue level, draw the sample 0.5 - 2.0 hours after an infusion.3

Trough levels are reflected by samples obtained immediately prior to the next dose.9

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Type:

Serum or plasma is the recommended specimen. Whole blood cannot be used. The anticoagulants EDTA, sodium heparin, lithium 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.

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Handling Conditions:

Vancomycin samples are generally considered stable for 3 days when stored refrigerated at 2 - 8C and stable for 1 month when stored frozen < -20C.9

Preferably, blood specimens should be separated and tested immediately after collection, or separated specimens should be frozen. Thaw frozen specimens and test them immediately.

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. It is recommended that human specimens be handled in accordance with the OSHA Standard on Bloodborne Pathogens or other appropriate local practices.5,6

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EQUIPMENT AND MATERIALS:

Equipment:

Beckman Coulter AU400/AU400e, AU480, AU600, AU640/AU640e, AU680, AU2700, and AU5400 analyzers.

Materials:

Emit( 2000 Vancomycin Assay

Enzyme Reagent 1 - Vancomycin labeled with bacterial G6PDH, HEPES buffer, bovine serum albumin, stabilizers, and preservatives.

Antibody/Substrate Reagent 2 - mouse monoclonal antibodies reactive to vancomycin, G6P, NAD, bovine serum albumin, stabilizers, and preservatives.

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Test tubes 12 -16 mm in diameter or sample cups (Cat No. AU1063)

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Emit( 2000 Vancomycin Calibrators (Cat No. 4W109)

The Emit( Vancomycin Calibrators contain the following stated vancomycin concentrations: 0 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 30 μg/mL, 50 μg/mL. The calibrators also contain buffer, stabilizers, preservatives, and 0.09% sodium azide.

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Preparation

The Emit( 2000 Vancomycin Calibrators are provided ready to use and may be used directly from the refrigerator. No reconstitution is necessary.

Emit( 2000 Vancomycin 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 Vancomycin Assay and Calibrators are for in vitro diagnostic use.

2. 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 with local regulations.

4. Do not use the reagent kit or calibrators after the expiration date.

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.5,6

Storage and Stability:

Unopened Emit® 2000 Vancomycin 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 Vancomycin 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.

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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

Within run precision was calculated according to Clinical and Laboratory Standards Institute (CLSI EP5-A) by running two replicates of each control level twice daily for twenty days (N=80). Total precision was calculated from this data.

| |Within Run Precision |Total Precision |

| |Level 1 |

|Intercept ((g/mL) |-0.227 |

|Correlation Coefficient |0.997 |

|Number of Samples |62 |

CALIBRATION:

Perform a multi-point calibration (5AB) using a water blank (blue rack) and the Emit® 2000 Vancomycin Calibrators: 5, 10, 20, 30, 50. 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 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 .

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 vancomycin, multiply by 0.67.

REPORTING RESULTS:

Reference Ranges:

Reported peak therapeutic ranges for vancomycin vary considerably. Both the dosage and the timing of sample collection may affect the peak therapeutic range. 1,3,7,8 For example, after completion of a 60-minute infusion of vancomycin in adults, samples drawn at 2 hours had concentrations of 18 - 26 μg/mL (12 - 17 μmol/L),3 samples drawn at 1 hour had concentrations of 26.5 - 40 μg/mL (18 - 27 μmol/L),8 and samples drawn at 30 minutes had concentrations of 30 - 40 μg/mL (20 - 27 μmol/L),1-3

Trough vancomycin serum concentrations of 5.0 – 10 μg/mL (3.4 – 6.7 μmol/L) usually ensure that the concentration is above the minimum inhibitory concentrations of most vancomycin-sensitive pathogens and that the drug elimination is adequate. 1,7

For patients on concomitant vancomycin and aminoglycoside treatment, peak vancomycin concentrations exceeding 30 μg/mL (20 μmol/L) and trough concentrations above 10 μg/mL (6.7 μmol/L) are associated with nephrotoxicity. 1 Serum concentrations above 80 μg/mL (54 μmol/L) are associated with ototoxicity.1

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.

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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

For purposes of diagnosis and treatment, results of this test should always be interpreted in conjunction with the patient’s medical history, clinical presentation and other findings.

The concentration of vancomycin 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 variations in absorption, distribution, biotransformation, and excretion. These parameters must be considered when interpreting results.1

The factors that can influence the relationship between vancomycin serum or plasma concentrations and clinical response include renal function, concomitant drug therapy, susceptibility of the infecting organism to vancomycin, severity of infection, general state of health and use of other drugs.1

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LIMITATIONS:

The Emit® 2000 Vancomycin Assay accurately quantitates vancomycin concentrations in human serum or plasma containing 2.0 - 50 g/mL

(3.4 - 34 mol/L) vancomycin.

To estimate vancomycin concentrations above the assay range, patient samples containing more than 50 g/mL (34 mol/L) vancomycin may be diluted with one or two parts distilled or deionized water or Emit® 2000 Vancomycin 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 20 (g/mL vancomycin and either 400 mg/dL hemoglobin, 750 mg/dL triglycerides, or 30 mg/dL bilirubin were added to simulate hemolytic, lipemic, or icteric samples. Grossly hemolyzed samples should be recollected.

Sensitivity

The sensitivity level of the Emit( 2000 Vancomycin Assay is 2.0 g/mL. This level represents the lowest concentration of vancomycin that can be distinguished from 0 g/mL with a confidence level of 95%.

Specificity

The Emit( 2000 Vancomycin Assay measures the total (protein-bound plus unbound) vancomycin 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 table on the following page do not interfere with the Emit( 2000 Vancomycin Assay when tested in the presence of 20g/mL vancomycin. Levels tested were at or above maximum physiological or pharmacological concentrations.

|Compound |Concentration Tested |Compound |Concentration Tested |

| |((g/mL) | |((g/mL) |

|Acyclovir |25 |Furosemide |100 |

|Amikacin |100 |Fusidic Acid |500 |

|Amphotericin B |20 |Gentamicin |100 |

|Aztreonam |200 |Imipenem |70 |

|Caffeine |2 |Methicillin |500 |

|CDP-1 |20 |Metronidazole |50 |

|Cefazoline |500 |Netilmicin |100 |

|Cefotaxine |1000 |Nitroprusside |60 |

|Chloramphenicol |100 |Penicillin G* |10 |

|Ciprofloxicin |10 |Pentamidine |0.7 |

|Cisplatin |25 |Phenobarbital |40 |

|Clindamycin |10 |Rifampin |500 |

|Cyclosporine |50 |Salicylate |60 |

|Digoxin |0.006 |Sulphamethoxazole |600 |

|Epinephrine |1 |Theophylline |20 |

|Erythromycin |5 |Trimethoprim |25 |

|Ethacrynic Acid |50 |Tobramycin |100 |

|Flucytosine |100 | | |

* Approximately equivalent to 16.7 units/mL penicillin G.

REFERENCES:

1. Rodvold KA, Erdman SM, Pryka RD: Vancomycin, in Schumacher GE (ed): Therapeutic Drug Monitoring. Norwalk, CT: Appleton and Lange; 1995:587-632.

2. Hsu P, Ernst R, Levy M: Emit® 2000 tobramycin and vancomycin assays [abstract]. Clin Chem 2000; 46(suppl 6):page A195. Abstract 762.

3. Fitzsimmons WE, Postelnick MJ, Tortorice PV: Survey of vancomycin monitoring guidelines in Illinois hospitals. Drug Intell Clin Pharm. 1988;22:598-600.

4. Polk RE, Espinel-Ingroff A, Lockridge R: In vitro evaluation of a vancomycin radioimmunoassay and observations on vancomycin pharmacokinetics in dialysis patients. Drug Intell Clin Pharm. 1981;15:15-20.

5. 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.

6. World Health Organization. Laboratory Biosafety Manual. 2nd Ed. Geneva: World Health Organization; 1993.

7. Moyer TP: Therapeutic drug monitoring, in Burtis CA, Ashwood ER (eds): Tietz Textbook of Clinical Chemistry. 3rd ed. Philadelphia, Pennsylvania: WB Saunders; 1999:886-905.

8. Healy DP, Polk RE, Garson ML, Rock DT, Comstock TJ. Comparison of steady-state pharmacokinetics of two dosage regimens of vancomycin in normal volunteers. Antimicrob Agents Chemother. 1987;31(3):393-397.

9. CAP Patient Prep & Specimen Handling Fascicle IV: Therapeutic Drug Monitoring/Toxicology. Committee on Patient Preparation and Specimen Handling. 1985. Aminoglycosides.

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